Sugar and lipid metabolism regulators in plants iv

ABSTRACT

Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation are provided. In particular, lipid metabolism proteins (LMP) and encoding nucleic acids originating from  Arabidopsis thaliana, Brassica napus , and  Physcomitrella patens  are provided. The nucleic acids and proteins are used in methods of producing transgenic plants and modulating levels of seed storage compounds. Preferably, the seed storage compounds are lipids, fatty acids, starches, or seed storage proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/943,078 filed Nov. 10, 2010, which is a divisional application ofU.S. application Ser. No. 10/523,503 filed Jul. 13, 2005, which is anational stage application (under 35 U.S.C. §371) of PCT/US2003/24364filed Aug. 4, 2003, which claims benefit U.S. Provisional PatentApplication Ser. No. 60/400,803 filed Aug. 2, 2002. The entire contentsof each of these applications are hereby incorporated by referenceherein.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_List_(—)13987_(—)00179_US. The size ofthe text file is 207 KB, and the text file was created on Apr. 25, 2012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingproteins that are related to the presence of seed storage compounds inplants. More specifically, the present invention relates to nucleic acidsequences encoding sugar and lipid metabolism regulator proteins and theuse of these sequences in transgenic plants. The invention furtherrelates to methods of applying these novel plant polypeptides to theidentification and stimulation of plant growth and/or to the increase ofyield of seed storage compounds.

2. Background Art

The study and genetic manipulation of plants has a long history thatbegan even before the framed studies of Gregor Mendel. In perfectingthis science, scientists have accomplished modification of particulartraits in plants ranging from potato tubers having increased starchcontent to oilseed plants such as canola and sunflower having increasedor altered fatty acid content. With the increased consumption and use ofplant oils, the modification of seed oil content and seed oil levels hasbecome increasingly widespread (e.g. Töpfer et al., 1995, Science268:681-686). Manipulation of biosynthetic pathways in transgenic plantsprovides a number of opportunities for molecular biologists and plantbiochemists to affect plant metabolism giving rise to the production ofspecific higher-value products. The seed oil production or compositionhas been altered in numerous traditional oilseed plants such as soybean(U.S. Pat. No. 5,955,650), canola (U.S. Pat. No. 5,955,650), sunflower(U.S. Pat. No. 6,084,164), rapeseed (Töpfer et al., 1995, Science268:681-686), and non-traditional oil seed plants such as tobacco(Cahoon et al., 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).

Plant seed oils comprise both neutral and polar lipids (See Table 1).The neutral lipids contain primarily triacylglycerol, which is the mainstorage lipid that accumulates in oil bodies in seeds. The polar lipidsare mainly found in the various membranes of the seed cells, e.g. theendoplasmic reticulum, microsomal membranes, and the cell membrane. Theneutral and polar lipids contain several common fatty acids (See Table2) and a range of less common fatty acids. The fatty acid composition ofmembrane lipids is highly regulated and only a select number of fattyacids are found in membrane lipids. On the other hand, a large number ofunusual fatty acids can be incorporated into the neutral storage lipidsin seeds of many plant species (Van de Loo F. J. et al., 1993, UnusualFatty Acids in Lipid Metabolism in Plants pp. 91-126, editor T S MooreJr. CRC Press; Millar et al., 2000, Trends Plant Sci. 5:95-101).

TABLE 1 Plant Lipid Classes Neutral Lipids Triacylglycerol (TAG)Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar LipidsMonogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG)Phosphatidylglycerol (PG) Phosphatidylcholine (PC)Phosphatidylethanolamine (PE) Phosphatidylinositol (PI)Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol

TABLE 2 Common Plant Fatty Acids 16:0 Palmitic acid 16:1 Palmitoleicacid 16:3 Palmitolenic acid 18:0 Stearic acid 18:1 Oleic acid 18:2Linoleic acid 18:3 Linolenic acid γ-18:3   Gamma-linolenic acid* 20:0Arachidic acid 20:1 Eicosenoic acid 22:6 Docosahexanoic acid (DHA)* 20:2Eicosadienoic acid 20:4 Arachidonic acid (AA)* 20:5 Eicosapentaenoicacid (EPA)* 22:1 Erucic acid

In Table 2, the fatty acids denoted with an asterisk do not normallyoccur in plant seed oils, but their production in transgenic plant seedoil is of importance in plant biotechnology.

Lipids are synthesized from fatty acids, and their synthesis may bedivided into two parts: the prokaryotic pathway and the eukaryoticpathway (Browse et al., 1986, Biochemical J. 235:25-31; Ohlrogge &Browse, 1995, Plant Cell 7:957-970). The prokaryotic pathway is locatedin plastids, the primary site of fatty acid biosynthesis. Fatty acidsynthesis begins with the conversion of acetyl-CoA to malonyl-CoA byacetyl-CoA carboxylase (ACCase). Malonyl-CoA is converted tomalonyl-acyl carrier protein (ACP) by the malonyl-CoA:ACP transacylase.The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes acondensation reaction in which the acyl group from acetyl-CoA istransferred to malonyl-ACP to form 3-ketobutyryl-ACP. In a subsequentseries of condensation, reduction and dehydration reactions the nascentfatty acid chain on the ACP cofactor is elongated by the step-by-stepaddition (condensation) of two carbon atoms donated by malonyl-ACP untila 16-carbon or 18-carbon saturated fatty acid chain is formed. Theplastidial delta-9 acyl-ACP desaturase introduces the first unsaturateddouble bond into the fatty acid. Thioesterases cleave the fatty acidsfrom the ACP cofactor, and free fatty acids are exported to thecytoplasm where they participate as fatty acyl-CoA esters in theeukaryotic pathway. In the eukaryotic pathway, the fatty acids areesterified by glycerol-3-phosphate acyltransferase and lysophosphatidicacid acyltransferase to the sn-1 and sn-2 positions ofglycerol-3-phosphate, respectively, to yield phosphatidic acid (PA). ThePA is the precursor for other polar and neutral lipids, the latter beingformed in the Kennedy pathway (Voelker, 1996, Genetic Engineeringed.:Setlow 18:111-113; Shanklin & Cahoon, 1998, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 49:611-641; Frentzen, 1998, Lipids100:161-166; Millar et al., 2000, Trends Plant Sci. 5:95-101).

Storage lipids in seeds are synthesized from carbohydrate-derivedprecursors. Plants have a complete glycolytic pathway in the cytosol(Plaxton, 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185-214),and it has been shown that a complete pathway also exists in theplastids of rapeseeds (Kang & Rawsthorne, 1994, Plant J. 6:795-805).Sucrose is the primary source of carbon and energy, transported from theleaves into the developing seeds. During the storage phase of seeds,sucrose is converted in the cytosol to provide the metabolic precursorsglucose-6-phosphate and pyruvate. These are transported into theplastids and converted into acetyl-CoA that serves as the primaryprecursor for the synthesis of fatty acids. Acetyl-CoA in the plastidsis the central precursor for lipid biosynthesis. Acetyl-CoA can beformed in the plastids by different reactions, and the exactcontribution of each reaction is still being debated (Ohlrogge & Browse,1995, Plant Cell 7:957-970). It is accepted, however, that a large partof the acetyl-CoA is derived from glucose-6-phospate and pyruvate thatare imported from the cytoplasm into the plastids. Sucrose is producedin the source organs (leaves, or anywhere that photosynthesis occurs)and is transported to the developing seeds that are also termed sinkorgans. In the developing seeds, the sucrose is the precursor for allthe storage compounds, i.e. starch, lipids and partly the seed storageproteins. Therefore, it is clear that carbohydrate metabolism in whichsucrose plays a central role is very important to the accumulation ofseed storage compounds.

Although lipid and fatty acid content of seed oil can be modified by thetraditional methods of plant breeding, the advent of recombinant DNAtechnology has allowed for easier manipulation of the seed oil contentof a plant, and in some cases, has allowed for the alteration of seedoils in ways that could not be accomplished by breeding alone (See,e.g., Töpfer et al. 1995, Science 268:681-686). For example,introduction of a Δ¹²-hydroxylase nucleic acid sequence into transgenictobacco resulted in the introduction of a novel fatty acid, ricinoleicacid, into the tobacco seed oil (Van de Loo et al., 1995, Proc. Natl.Acad. Sci. USA 92:6743-6747). Tobacco plants have also been engineeredto produce low levels of petroselinic acid by the introduction andexpression of an acyl-ACP desaturase from coriander (Cahoon et al.,1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).

The modification of seed oil content in plants has significant medical,nutritional, and economic ramifications. With regard to the medicalramifications, the long chain fatty acids (C18 and longer) found in manyseed oils have been linked to reductions in hypercholesterolemia andother clinical disorders related to coronary heart disease (Brenner,1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a planthaving increased levels of these types of fatty acids may reduce therisk of heart disease. Enhanced levels of seed oil content also increaselarge-scale production of seed oils and thereby reduce the cost of theseoils.

In order to increase or alter the levels of compounds such as seed oilsin plants, nucleic acid sequences and proteins regulating lipid andfatty acid metabolism must be identified. As mentioned earlier, severaldesaturase nucleic acids such as the Δ⁶-desaturase nucleic acid,Δ¹²-desaturase nucleic acid and acyl-ACP desaturase nucleic acid havebeen cloned and demonstrated to encode enzymes required for fatty acidsynthesis in various plant species. Oleosin nucleic acid sequences fromsuch different species as Brassica, soybean, carrot, pine, andArabidopsis thaliana have also been cloned and determined to encodeproteins associated with the phospholipid monolayer membrane of oilbodies in those plants.

It has also been determined that two phytohormones, gibberellic acid(GA) and absisic acid (ABA), are involved in overall regulatoryprocesses in seed development (e.g. Ritchie & Gilroy, 1998, PlantPhysiol. 116:765-776; Arenas-Huertero et al., 2000, Genes Dev.14:2085-2096). Both the GA and ABA pathways are affected by okadaicacid, a protein phosphatase inhibitor (Kuo et al., 1996, Plant Cell.8:259-269). The regulation of protein phosphorylation by kinases andphosphatases is accepted as a universal mechanism of cellular control(Cohen, 1992, Trends Biochem. Sci. 17:408-413). Likewise, the planthormones ethylene (e.g. Zhou et al., 1998, Proc. Natl. Acad. Sci. USA95:10294-10299; Beaudoin et al., 2000, Plant Cell 2000:1103-1115), andauxin (e.g. Colon-Caimona et al., 2000, Plant Physiol. 124:1728-1738)are involved in controlling plant development as well.

Although several compounds are known that generally affect plant andseed development, there is a clear need to specifically identify factorsthat are more specific for the developmental regulation of storagecompound accumulation and to identify genes which have the capacity toconfer altered or increased oil production to its host plant and toother plant species. This invention discloses a large number of nucleicacid sequences from Arabidopsis thaliana, Brassica napus, and the mossPhyscomitrella patens. These nucleic acid sequences can be used to alteror increase the levels of seed storage compounds such as proteins,sugars and oils, in plants, including transgenic plants, such asrapeseed, canola, linseed, soybean, sunflower maize, oat, rye, barley,wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor andpeanut, which are oilseed plants containing high amounts of lipidcompounds.

SUMMARY OF THE INVENTION

The present invention provides novel isolated nucleic acid and aminoacid sequences associated with the metabolism of seed storage compoundsin plants.

The present invention also provides an isolated nucleic acid fromArabidopsis, Brassica, and Physcomitrella patens encoding a LipidMetabolism Protein (LMP), or a portion thereof. These sequences may beused to modify or increase lipids and fatty acids, cofactors and enzymesin microorganisms and plants.

Arabidopsis plants are known to produce considerable amounts of fattyacids such as linoleic and linolenic acid (See, e.g., Table 2) and fortheir close similarity in many aspects (gene homology, etc.) to the oilcrop plant Brassica. Therefore, nucleic acid molecules originating froma plant like Arabidopsis thaliana and Brassica napus are especiallysuited to modify the lipid and fatty acid metabolism in a host,especially in microorganisms and plants. Furthermore, nucleic acids fromthe plants Arabidopsis thaliana and Brassica napus can be used toidentify those DNA sequences and enzymes in other species which areuseful to modify the biosynthesis of precursor molecules of fatty acidsin the respective organisms.

The present invention further provides an isolated nucleic acidcomprising a fragment of at least 15 nucleotides of a nucleic acid froma plant (Arabidopsis thaliana, Brassica napus, or Physcomitrella patens)encoding a Lipid Metabolism Protein (LMP), or a portion thereof.

Also provided by the present invention are polypeptides encoded by thenucleic acids, heterologous polypeptides comprising polypeptides encodedby the nucleic acids, and antibodies to those polypeptides.

Additionally, the present invention relates to and provides the use ofLMP nucleic acids in the production of transgenic plants having amodified level of a seed storage compound. A method of producing atransgenic plant with a modified level of a seed storage compoundincludes the steps of transforming a plant cell with an expressionvector comprising a LMP nucleic acid, and generating a plant with amodified level of the seed storage compound from the plant cell. In apreferred embodiment, the plant is an oil producing species selectedfrom the group consisting of rapeseed, canola, linseed, soybean,sunflower, maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oilpalm, coconut palm, flax, castor, and peanut, for example.

According to the present invention, the compositions and methodsdescribed herein can be used to increase or decrease the level of an LMPin a transgenic plant comprising increasing or decreasing the expressionof the LMP nucleic acid in the plant. Increased or decreased expressionof the LMP nucleic acid can be achieved through in vivo mutagenesis ofthe LMP nucleic acid. The present invention can also be used to increaseor decrease the level of a lipid in a seed oil, to increase or decreasethe level of a fatty acid in a seed oil, or to increase or decrease thelevel of a starch in a seed or plant.

Also included herein is a seed produced by a transgenic planttransformed by a LMP DNA sequence, wherein the seed contains the LMP DNAsequence and wherein the plant is true breeding for a modified level ofa seed storage compound. The present invention additionally includes aseed oil produced by the aforementioned seed.

Further provided by the present invention are vectors comprising thenucleic acids, host cells containing the vectors, and descendent plantmaterials produced by transforming a plant cell with the nucleic acidsand/or vectors.

According to the present invention, the compounds, compositions, andmethods described herein can be used to increase or decrease the levelof a lipid in a seed oil, or to increase or decrease the level of afatty acid in a seed oil, or to increase or decrease the level of astarch or other carbohydrate in a seed or plant. A method of producing ahigher or lower than normal or typical level of storage compound in atransgenic plant, comprises expressing a LMP nucleic acid fromArabidopsis thaliana, Brassica napus, and Physcomitrella patens in thetransgenic plant, wherein the transgenic plant is Arabidopsis thalianaand Brassica napus, or a species different from Arabidopsis thaliana andBrassica napus. Also included herein are compositions and methods of themodification of the efficiency of production of a seed storage compound.As used herein, the phrase “Arabidopsis thaliana and Brassica napus”means Arabidopsis thaliana and/or Brassica napus.

Accordingly, the present invention provides novel isolated LMP nucleicacids and isolated LMP amino acid sequences from Arabidopsis thaliana,Brassica napus, and Physcomitrella patens, as well as active fragments,analogs and orthologs thereof.

The present invention also provides transgenic plants having modifiedlevels of seed storage compounds, and in particular, modified levels ofa lipid, a fatty acid, or a sugar.

The polynucleotides and polypeptides of the present invention, includingagonists and/or fragments thereof, also have uses that includemodulating plant growth, and potentially plant yield, preferablyincreasing plant growth under adverse conditions (drought, cold, light,UV). In addition, antagonists of the present invention may have usesthat include modulating plant growth and/or yield, preferably throughincreasing plant growth and yield. In yet another embodiment,overexpression of the polypeptides of the present invention using aconstitutive promoter (e.g., 35S or other promoters) may be useful forincreasing plant yield under stress conditions (drought, light, cold,UV) by modulating light utilization efficiency.

The present invention also provides methods for producing suchaforementioned transgenic plants. In another embodiment, the presentinvention provides seeds and seed oils from such aforementionedtransgenic plants.

These and other embodiments, features, and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included therein.

Before the present compounds, compositions, and methods are disclosedand described, it is to be understood that this invention is not limitedto specific nucleic acids, specific polypeptides, specific cell types,specific host cells, specific conditions, or specific methods, etc., assuch may, of course, vary, and the numerous modifications and variationstherein will be apparent to those skilled in the art. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. As used in the specification and in the claims, “a” or “an”can mean one or more, depending upon the context in which it is used.Thus, for example, reference to “a cell” can mean that at least one cellcan be utilized.

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides anisolated nucleic acid from a plant (Arabidopsis thaliana, Brassicanapus, and Physcomitrella patens) encoding a Lipid Metabolism Protein(LMP), or a portion thereof. As used herein, the phrase “Arabidopsisthaliana, Brassica napus, and Physcomitrella patens” means Arabidopsisthaliana and/or Brassica napus and/or Physcomitrella patens.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode LMP polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification of anLMP-encoding nucleic acid (e.g., LMP DNA). As used herein, the terms“nucleic acid molecule” and “polynucleotide sequence” are usedinterchangeably and are intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of a gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is substantially separated from other nucleic acidmolecules which are present in the natural source of the nucleic acid.Preferably, an “isolated” nucleic acid is substantially free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated LMP nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived (e.g., anArabidopsis thaliana or Brassica napus cell). Moreover, an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or chemical precursors, or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a polynucleotide sequence of Appendix A (i.e. thepolynucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, or SEQ ID NO:81, or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. For example, an Arabidopsis thaliana, Brassica napus,or Physcomitrella patens LMP cDNA can be isolated from an Arabidopsisthaliana, Brassica napus, or Physcomitrella patens library using all orportion of one of the polynucleotide sequences of Appendix A as ahybridization probe and standard hybridization techniques (e.g., asdescribed in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Moreover, a nucleic acidmolecule encompassing all or a portion of one of the polynucleotidesequences of Appendix A can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of Appendix A can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this same sequence ofAppendix A). For example, mRNA can be isolated from plant cells (e.g.,by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.,1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the polynucleotide sequences shown inAppendix A. A nucleic acid of the invention can be amplified using cDNAor, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a LMP nucleotide sequencecan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid of the inventioncomprises one of the polynucleotide sequences shown in Appendix A (i.e.SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81). Thesepolynucleotides of Appendix A correspond to the Arabidopsis thaliana,Brassica napus, and Physcomitrella patens LMP cDNAs of the invention.These cDNAs comprise sequences encoding LMPs (i.e., the “coding region”or open reading frame (ORF)), as well as 5′ untranslated sequences and3′ untranslated sequences. Alternatively, the nucleic acid molecules cancomprise only the coding region of any of the polynucleotide sequencesdescribed herein or can contain whole genomic fragments isolated fromgenomic DNA.

For the purposes of this application, it will be understood that each ofthe polynucleotide sequences set forth in Appendix A has an identifyingentry number (e.g., Pk123). Each of these sequences may generallycomprise three parts: a 5′ upstream region, a coding region, and adownstream region. The particular polynucleotide sequences shown inAppendix A represent the coding region or open reading frame, and theputative functions of the encoded polypeptides are indicated in Table 3.

TABLE 3 Putative LMP Functions Sequence SEQ ID code Function NO: Pk123Gibberellin-regulated protein 1 GASA3 precursor Pk197 Tyrosineaminotransferase 3 Pk136 D-hydroxy-fatty acid dehydrogenase 5 Pk156Serine protease 7 Pk159 Nonspecific lipid-transfer protein 9 Pk179Signal transduction protein 11 Pk202 Lipid transfer - like protein 13Pk206 bZIP transcription factor 15 Pk207 Acyl-CoA dehydrogenase 17 Pk209Pyruvate kinase 19 Pk215 Phosphatidylglycerotransferase 21 Pk239Digalactosyldiacylglycerol 23 synthase Pk240 Phosphatidatecytidyltransferase 25 Pk241 AT Psbs protein 27 Pk242 Omega-6 fatty aciddesaturase, 29 endoplasmic reticulum (FAD2) Bn011 Gibberellin 3-betahydroxylase 31 with +4 G Bn077 Zinc finger DNA binding protein 33 Jb001Gibberellin 20-oxidase 35 Jb002 Seed maturation protein 37 Jb003Beta-VPE Vacuolar Processing Enzym 39 Jb005 Very-long-chain fatty acid41 condensing enzyme CUT1 Jb007 Glucokinase 43 Jb009 GlutathioneS-transferase TSI-1 45 Jb013 ABA-regulated gene 47 Jb017 Cysteineproteinase 51 Jb024 Pectinesterase-like protein 53 Jb027 Signaltransduction protein 55 OO-1 Aldose reductase-like protein 57 OO-2Dormancy related protein 59 OO-3 HSP associated protein like 61 OO-4Poly (ADP-ribose) polymerase 63 OO-5 Transitional endoplasmic 65reticulum ATPase OO-6 Beta coat like protein 67 OO-8 Proteindisulfide-isomerase 69 OO-9 Signal transduction protein/ 71 Apoptosisinhibitor OO-10 Annexin 73 OO-11 Putative oxidoreductase 75 OO-12 Longchain alc dehydrogenase/ 77 oxidoreductase pp82 Transcription factor 79Pk225 Amino-cyclopropane-carboxylic 81 acid oxidase

TABLE 4 Grouping of LMPs based on Functional protein domains FunctionalSEQ SEQ Domain category ID: Code: Functional domain position DNA-binding1 Pk123 Zinc finger 66-86 proteins 29-71 15 Pk206 bZIP transcriptionfactor (PFAM) 144-197 Leucine zipper 179-209 27 Pk241 DNA-binding domain207-221 Histone H5 signature 57-71 33 Bn077 Zinc finger (BRCT; PARP) 64-104 Ethylene responsive element binding protein 79-99 63 OO-4 Zincfinger 760-805 Leucine zipper 114-117 73 OO-10 Zinc finger 220-230 YeastDNA-binding domain 207-217 79 pp82 Myb DNA-binding domain  19-119Kinases 43 Jb007 Glucokinase 173-206 45 Jb009 Deoxynucleoside kinase 99-139 19 Pk209 Pyruvate kinase (PFAM)   1-326 61 OO-3 Galactokinase285-296 Signal 67 OO-6 Wnt-1 domain 607-655 Transduction WSC domain527-548 71 OO-9 BIR repeat (inhibitor of apoptosis) 47-85 Wnt-1 domain43-91 41 Jb005 Wnt-1 domain 23-71 47 Jb013 Wnt-1 domain 23-91 55 Jb027Emp24/gp25L intracellular vesicle trafficking   2-204 Wnt-1 domain135-183 11 Pk179 Wnt-1 domain 279-327 PDZ domain (Wnt signalling)205-299 3 Pk197 Wnt-1 domain 300-348 Proteases 7 Pk156 Serine protease171-191 Prolyl aminopeptidase 128-139 37 Jb002 Peptidase family M23/M37404-444 39 Jb003 Cysteine protease 52-76 Peptidase C13 (PFAM)  10-367 51Jb017 Cysteine protease C1 163-178 Peptidase C1 (PFAM) 145-361 65 OO-5Peptidase family M41 343-387 620-664 AAA ATPase molecular chaperone(PFAM) 243-427 Lipid 5 Pk136 D-Hydroxy-fatty acid dehydrogenase  94-143metabolism 9 Pk159 Lipid Transfer Protein LTP (PFAM)  29-117 13 Pk202Lipid Transfer Protein LTP (PFAM)  38-103 17 Pk207 Acyl-CoAdehydrogenase  2-44 Iron-containing alcohol dehydrogenase  97-112 21Pk215 CDP-alcohol phosphatidyltransferase (PFAM) 172-309 23 Pk239Glycosyl (galactosyl) transferase (PFAM) 572-674 25 Pk240 Phosphatidatecytidyltransferase 343-370 29 Pk242 Fatty acid desaturase (PFAM)  32-376Oxido- 31 Bn011 Iron Ascorbate oxidoreductase (PFAM)  43-343 reductases35 Jb001 Respiratory chain NADH dehydrogenase  95-123 Iron Ascorbateoxidoreductase (PFAM)  54-369 53 Jb024 Multicopper oxidase 216-247123-145 Copper-oxidase (PFAM) 154-306 57 OO-1 Aldo/keto reductase family(PFAM)  18-294 59 OO-2 Alcohol dehydrogenase (PFAM)  38-228 69 OO-8Thioredoxin (PFAM)  22-250 75 OO-11 Alcohol dehydrogenase (PFAM)  50-23477 OO-12 Zinc alcohol dehydrogenase(PFAM)  20-329 81 Pk225 IronAscorbate oxidoreductase (PFAM)   3-297

In another preferred embodiment, an isolated nucleic acid molecule ofthe present invention encodes a polypeptide that is able to participatein the metabolism of seed storage compounds such as lipids, starch, andseed storage proteins, and that contains a DNA-binding (or transcriptionfactor) domain, a protein kinase domain, a signal transduction domain, aprotease domain, a lipid metabolism domain, or an oxidoreductase domain.Examples of isolated nucleic acids that encode LMPs containing suchdomains can be found in Table 4. Examples of nucleic acids encoding LMPscontaining a DNA-binding domain include those shown in SEQ ID NO:1, SEQID NO:15, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:63, SEQ ID NO:73, andSEQ ID NO:79. Examples of nucleic acids encoding LMPs containing aprotein kinase domain include those shown in SEQ ID NO:19, SEQ ID NO:43,SEQ ID NO:45, and SEQ ID NO:61. Examples of nucleic acids encoding LMPscontaining a signal transduction domain include those shown in SEQ IDNO:3, SEQ ID NO:11, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:55, SEQ IDNO:67, and SEQ ID NO:71. Examples of nucleic acids encoding LMPscontaining a protease domain include those shown in SEQ ID NO:7, SEQ IDSEQ ID NO:39, SEQ ID NO:51, and SEQ ID NO:65. Examples of nucleic acidsencoding LMPs containing a lipid metabolism domain include those shownin SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:29. Examples of nucleic acidsencoding LMPs containing a oxidoreductase domain include those shown inSEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:81.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule, which is a complementof one of the polynucleotide sequences shown in Appendix A (i.e. SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81), or a portionthereof. A nucleic acid molecule which is complementary to one of thepolynucleotide sequences shown in Appendix A is one which issufficiently complementary to one of the polynucleotide sequences shownin Appendix A such that it can hybridize to one of the nucleotidesequences shown in Appendix A, thereby forming a stable duplex.

In another preferred embodiment, an isolated nucleic acid of theinvention comprises a polynucleotide sequence encoding a polypeptideselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, or SEQ ID NO:82.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a polynucleotide sequence which is at leastabout 50-60%, preferably at least about 60-70%, more preferably at leastabout 70-80%, 80-90%, or 90-95%, and even more preferably at least about95%, 96%, 97%, 98%, 99%, or more homologous to a polynucleotide sequenceshown in Appendix A, or a portion thereof. In an additional preferredembodiment, an isolated nucleic acid molecule of the invention comprisesa polynucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to one of the polynucleotide sequences shown inAppendix A, or a portion thereof. These stringent conditions includewashing with a solution having a salt concentration of about 0.02 M atpH 7 and about 60° C. In another embodiment, the stringent conditionscomprise an initial hybridization in a 6× sodium chloride/sodium citrate(6×SSC) solution at 65° C.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in Appendix A, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of a LMP. The polynucleotidesequences determined from the cloning of the LMP genes from Arabidopsisthaliana, Brassica napus, and Physcomitrella patens allows for thegeneration of probes and primers designed for use in identifying and/orcloning LMP homologues in other cell types and organisms, as well as LMPhomologues from other plants or related species. Therefore thisinvention also provides compounds comprising the nucleic acids disclosedherein, or fragments thereof. These compounds include the nucleic acidsattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like.The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50, or 75consecutive nucleotides of a sense strand of one of the sequences setforth in Appendix A, an anti-sense sequence of one of the sequences setforth in Appendix A, or naturally occurring mutants thereof. Primersbased on a polynucleotide sequence of Appendix A can be used in PCRreactions to clone LMP homologues. Probes based on the LMP nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous proteins. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g. the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a genomic markertest kit for identifying cells which express a LMP, such as by measuringa level of a LMP-encoding nucleic acid in a sample of cells, e.g.,detecting LMP mRNA levels or determining whether a genomic LMP gene hasbeen mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid encoded by a sequence ofAppendix A such that the protein or portion thereof maintains the sameor a similar function as the wild-type protein. As used herein, thelanguage “sufficiently homologous” refers to proteins or portionsthereof which have amino acid sequences which include a minimum numberof identical or equivalent amino acid residues to an amino acid sequencesuch that the protein or portion thereof is able to participate in themetabolism of compounds necessary for the production of seed storagecompounds in plants, construction of cellular membranes inmicroorganisms or plants, or in the transport of molecules across thesemembranes. As used herein, an “equivalent” amino acid residue is, forexample, an amino acid residue which has a similar side chain as aparticular amino acid residue that is encoded by a polynucleotidesequence of Appendix A. Regulatory proteins, such as DNA bindingproteins, transcription factors, kinases, phosphatases, or proteinmembers of metabolic pathways such as the lipid, starch and proteinbiosynthetic pathways, or membrane transport systems, may play a role inthe biosynthesis of seed storage compounds. Examples of such activitiesare described herein (see putative annotations in Table 3). Examples ofLMP-encoding nucleic acid sequences are set forth in Appendix A.

As altered or increased sugar and/or fatty acid production is a generaltrait wished to be inherited into a wide variety of plants like maize,wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton,rapeseed, canola, manihot, pepper, sunflower and tagetes, solanaceousplants like potato, tobacco, eggplant, and tomato, Vicia species, pea,alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oilpalm, coconut), perennial grasses, and forage crops, these crop plantsare also preferred target plants for genetic engineering as one furtherembodiment of the present invention. As used herein, a “forage crop”includes, but is not limited to, Wheatgrass, Canarygrass, Bromegrass,Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, BirdsfootTrefoil, Alsike Clover, Red Clover, and Sweet Clover.

Portions of proteins encoded by the LMP nucleic acid molecules of theinvention are preferably biologically active portions of one of theLMPs. As used herein, the term “biologically active portion of a LMP” isintended to include a portion, e.g., a domain/motif, of a LMP thatparticipates in the metabolism of compounds necessary for thebiosynthesis of seed storage lipids, or the construction of cellularmembranes in microorganisms or plants, or in the transport of moleculesacross these membranes, or has an activity as set forth in Table 3. Todetermine whether a LMP or a biologically active portion thereof canparticipate in the metabolism of compounds necessary for the productionof seed storage compounds and cellular membranes, an assay of enzymaticactivity may be performed. Such assay methods are well known to thoseskilled in the art, and as described in Example 14 of theExemplification.

Biologically active portions of a LMP include peptides comprising aminoacid sequences derived from the amino acid sequence of a LMP (e.g., anamino acid sequence encoded by a nucleic acid sequence of Appendix A(i.e. SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81)or the amino acid sequence of a protein homologous to an LMP, whichinclude fewer amino acids than a full length LMP or the full lengthprotein which is homologous to an LMP) and exhibit at least one activityof an LMP. Typically, biologically active portions (peptides, e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100, or more amino acids in length) comprise a domain or motifwith at least one activity of a LMP. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of a LMP include one or more selected domains/motifs orportions thereof having biological activity.

Additional nucleic acid fragments encoding biologically active portionsof a LMP can be prepared by isolating a portion of one of the sequences,expressing the encoded portion of the LMP or peptide (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the LMP or peptide.

The invention further encompasses nucleic acid molecules that differfrom one of the polynucleotide sequences shown in Appendix A (i.e. SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81), andportions thereof) due to degeneracy of the genetic code and thus encodethe same LMP as that encoded by the polynucleotide sequences shown inAppendix A. In a further embodiment, the nucleic acid molecule of theinvention encodes a full length protein which is substantiallyhomologous to an amino acid sequence shown in Appendix A. In oneembodiment, the full-length nucleic acid or protein or fragment of thenucleic acid or protein is from Arabidopsis thaliana, Brassica napus,and Physcomitrella patens.

In addition to the Arabidopsis thaliana, Brassica napus, andPhyscomitrella patens LMP polynucleotide sequences described herein, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of LMPsmay exist within a population (e.g., the Arabidopsis thaliana, andBrassica napus, and Physcomitrella patens population). Such geneticpolymorphism in the LMP gene may exist among individuals within apopulation due to natural variation. As used herein, the terms “gene”and “recombinant gene” refer to nucleic acid molecules comprising anopen reading frame encoding a LMP, preferably an Arabidopsis thaliana,Brassica napus, or Physcomitrella patens LMP. Such natural variationscan typically result in 1-40% variance in the nucleotide sequence of theLMP gene. Any and all such nucleotide variations and resulting aminoacid polymorphisms in LMP that are the result of natural variation andthat do not alter the functional activity of LMPs are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural variants andnon-Arabidopsis thaliana and Brassica napus orthologs of the Arabidopsisthaliana, Brassica napus, and Physcomitrella patens LMP cDNA of theinvention can be isolated based on their homology to Arabidopsisthaliana, Brassica napus, and Physcomitrella patens LMP nucleic aciddisclosed herein using the Arabidopsis thaliana, Brassica napus, andPhyscomitrella patens cDNA, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions. As used herein, the term “orthologs” refers totwo nucleic acids from different species, but that have evolved from acommon ancestral gene by speciation. Normally, orthologs encode proteinshaving the same or similar functions. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a polynucleotide sequence shownin Appendix A. In other embodiments, the nucleic acid is at least 30,50, 100, 250, or more nucleotides in length. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75%, or more homologous to eachother typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65 C. In another embodiment, the stringentconditions comprise an initial hybridization in a 6× sodiumchloride/sodium citrate (6×SSC) solution at 65° C. Preferably, anisolated nucleic acid molecule of the invention that hybridizes understringent conditions to a polynucleotide sequence of Appendix A (i.e.SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81)corresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a polynucleotide sequence that occurs in nature(e.g., encodes a natural protein). In one embodiment, the nucleic acidencodes a natural Arabidopsis thaliana, Brassica napus, orPhyscomitrella patens LMP.

In addition to naturally-occurring variants of the LMP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a polynucleotidesequence of Appendix A, thereby leading to changes in the amino acidsequence of the encoded LMP, without altering the functional ability ofthe LMP. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in apolynucleotide sequence of Appendix A. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofone of the LMPs (Appendix A) without altering the activity of said LMP,whereas an “essential” amino acid residue is required for LMP activity.Other amino acid residues, however, (e.g., those that are not conservedor only semi-conserved in the domain having LMP activity) may not beessential for activity and thus are likely to be amenable to alterationwithout altering LMP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding LMPs that contain changes in amino acid residues thatare not essential for LMP activity. Such LMPs differ in amino acidsequence from a sequence yet retain at least one of the LMP activitiesdescribed herein. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50% homologous to anamino acid sequence encoded by a nucleic acid of Appendix A and iscapable of participation in the metabolism of compounds necessary forthe production of seed storage compounds in Arabidopsis thaliana,Brassica napus, and Physcomitrella patens, or cellular membranes, or hasone or more activities set forth in Table 3. Preferably, the proteinencoded by the nucleic acid molecule is at least about 50-60% homologousto one of the sequences encoded by a nucleic acid of Appendix A (i.e.SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, or SEQ ID NO:81), morepreferably at least about 60-70% homologous to one of the sequencesencoded by a nucleic acid of Appendix A, even more preferably at leastabout 70-80%, 80-90%, or 90-95% homologous to one of the sequencesencoded by a nucleic acid of Appendix A, and most preferably at leastabout 96%, 97%, 98%, or 99% homologous to one of the sequences encodedby a nucleic acid of Appendix A.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences encoded by a nucleic acid of Appendix A and a mutantform thereof) or of two nucleic acids, the sequences are aligned foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of one protein or nucleic acid for optimal alignment with theother protein or nucleic acid). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in one sequence (e.g., one of the sequencesencoded by a nucleic acid of Appendix A) is occupied by the same aminoacid residue or nucleotide as the corresponding position in the othersequence (e.g., a mutant form of the sequence encoded by a nucleic acidof Appendix A), then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=numbers ofidentical positions/total numbers of positions×100).

An isolated nucleic acid molecule encoding a LMP homologous to a proteinsequence encoded by a nucleic acid of Appendix A can be created byintroducing one or more nucleotide substitutions, additions, ordeletions into a polynucleotide sequence of Appendix A such that one ormore amino acid substitutions, additions, or deletions are introducedinto the encoded protein. Mutations can be introduced into one of thesequences of Appendix A by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted non-essential amino acid residue in a LMPis preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a LMP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a LMP activity described herein to identify mutants that retain LMPactivity. Following mutagenesis of one of the sequences of Appendix A,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined using, for example, assays describedherein (see Examples 13-14 of the Exemplification).

LMPs are preferably produced by recombinant DNA techniques. For example,a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described herein), and the LMP isexpressed in the host cell. The LMP can then be isolated from the cellsby an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, a LMP orpeptide thereof can be synthesized chemically using standard peptidesynthesis techniques. Moreover, native LMP can be isolated from cells,for example using an anti-LMP antibody, which can be produced bystandard techniques utilizing a LMP or fragment thereof of thisinvention.

The invention also provides LMP chimeric or fusion proteins. As usedherein, a LMP “chimeric protein” or “fusion protein” comprises a LMPpolypeptide operatively linked to a non-LMP polypeptide. An “LMPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a LMP, whereas a “non-LMP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the LMP, e.g., a protein whichis different from the LMP and which is derived from the same or adifferent organism. As used herein with respect to the fusion protein,the term “operatively linked” is intended to indicate that the LMPpolypeptide and the non-LMP polypeptide are fused to each other so thatboth sequences fulfill the proposed function attributed to the sequenceused. The non-LMP polypeptide can be fused to the N-terminus orC-terminus of the LMP polypeptide. For example, in one embodiment, thefusion protein is a GST-LMP (glutathione S-transferase) fusion proteinin which the LMP sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant LMPs. In another embodiment, the fusion protein is a LMPcontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofa LMP can be increased through use of a heterologous signal sequence.

Preferably, a LMP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (See, for example, Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnLMP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the LMP.

In addition to the nucleic acid molecules encoding LMPs described above,another aspect of the invention pertains to isolated nucleic acidmolecules which are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire LMP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a LMP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the entire coding region of Pk121 comprises nucleotides 1 to 786). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding LMP. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding LMP disclosed herein (e.g.,the polynucleotide sequences set forth in Appendix A), antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of LMP mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of LMP mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of LMP mRNA. An antisense oligonucleotide can be,for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotidesin length. An antisense or sense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylamino-methyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydro-uracil,beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyl-uracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diamino-purine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

In another variation of the antisense technology, a double-strandinterfering RNA construct can be used to cause a down-regulation of theLMP mRNA level and LMP activity in transgenic plants. This requirestransforming the plants with a chimeric construct containing a portionof the LMP sequence in the sense orientation fused to the antisensesequence of the same portion of the LMP sequence. A DNA linker region ofvariable length can be used to separate the sense and antisensefragments of LMP sequences in the construct.

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a LMP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic includingplant promoters are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an anomeric nucleic acid molecule. An anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNAin which, contrary to the usual units, the strands run parallel to eachother (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methyl-ribonucleotide (Inoue et al., 1987, Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff & Gerlach,1988, Nature 334:585-591)) can be used to catalytically cleave LMP mRNAtranscripts to thereby inhibit translation of LMP mRNA. A ribozymehaving specificity for an LMP-encoding nucleic acid can be designedbased upon the nucleotide sequence of an LMP cDNA disclosed herein(e.g., Pk123 in Appendix A) or on the basis of a heterologous sequenceto be isolated according to methods taught in this invention. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a LMP-encoding mRNA (See, e.g.,U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al.). Alternatively,LMP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules (See, e.g., Bartel,D. & Szostak J. W. 1993, Science 261:1411-1418).

Alternatively, LMP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a LMPnucleotide sequence (e.g., a LMP promoter and/or enhancers) to formtriple helical structures that prevent transcription of a LMP gene intarget cells (See generally, Helene C., 1991, Anticancer Drug Des.6:569-84; Helene C. et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J., 1992, Bioassays 14:807-15).

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a LMP (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. As used herein with respect to arecombinant expression vector, “operatively linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence and both sequences are fused to each other so that eachfulfills its proposed function (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers, and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruberand Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRCPress, Boca Raton, Fla., eds.: Glick & Thompson, Chapter 7, 89-108including the references therein. Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells or under certain conditions. It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., LMPs,mutant forms of LMPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of LMPs in prokaryotic or eukaryotic cells. For example, LMPgenes can be expressed in bacterial cells, insect cells (usingbaculovirus expression vectors), yeast and other fungal cells (SeeRomanos M. A. et al., 1992, Foreign gene expression in yeast: a review,Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al., 1991,Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, Bennet & Lasure, eds., p. 396-428: AcademicPress: an Diego; and van den Hondel & Punt, 1991, Gene transfer systemsand vector development for filamentous fungi, in: Applied MolecularGenetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge UniversityPress: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology1:239-251), ciliates of the types: Holotrichia, Peritrichia,Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, andStylonychia, especially of the genus Stylonychia lemnae with vectorsfollowing a transformation method as described in WO 98/01572, andmulticellular plant cells (See Schmidt & Willmitzer, 1988, Highefficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.:583-586;Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla.,chapter 6/7, S.71-119 (1993); White, Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,eds.: Kung and Wu, Academic Press 1993, 128-43; Potrykus, 1991, AnnuRev. Plant Physiol. Plant Mol. Biol. 42:205-225 (and references citedtherein)), or mammalian cells. Suitable host cells are discussed furtherin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. 1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve one or more of the following purposes: 1) to increase expressionof recombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith & Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. In one embodiment,the coding sequence of the LMP is cloned into a pGEX expression vectorto create a vector encoding a fusion protein comprising, from theN-terminus to the C-terminus, GST-thrombin cleavage site-X protein. Thefusion protein can be purified by affinity chromatography usingglutathione-agarose resin. Recombinant LMP unfused to GST can berecovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., 1990, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. 60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman S., 1990, GeneExpression Technology: Methods in Enzymology 185:119-128, AcademicPress, San Diego, Calif.). Another strategy is to alter the nucleic acidsequence of the nucleic acid to be inserted into an expression vector sothat the individual codons for each amino acid are those preferentiallyutilized in the bacterium chosen for expression (Wada et al., 1992,Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the LMP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, Embo J. 6:229-234), pMFa (Kuijan& Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel & Punt, 1991, “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy et al., eds., p. 1-28, Cambridge University Press:Cambridge.

Alternatively, the LMPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow & Summers, 1989, Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus, andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook,Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the LMPs of the invention may be expressed inuni-cellular plant cells (such as algae, see Falciatore et al. (1999,Marine Biotechnology 1:239-251 and references therein) and plant cellsfrom higher plants (e.g., the spermatophytes, such as crop plants).Examples of plant expression vectors include those detailed in: Becker,Kemper, Schell and Masterson (1992, “New plant binary vectors withselectable markers located proximal to the left border”, Plant Mol.Biol. 20:1195-1197) and Bevan (1984, “Binary Agrobacterium vectors forplant transformation, Nucleic Acids Res. 12:8711-8721; Vectors for GeneTransfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38).

A plant expression cassette preferably contains regulatory sequencescapable to drive gene expression in plant cells and which areoperatively linked so that each sequence can fulfil its function such astermination of transcription, including polyadenylation signals.Preferred polyadenylation signals are those originating fromAgrobacterium tumefaciens t-DNA such as the gene 3 known as octopinesynthase of the Ti-plasmid pTiACHS (Gielen et al. 1984, EMBO J. 3:835)or functional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptionallevels a plant expression cassette preferably contains other operativelylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallic et al.1987, Nucleic Acids Res. 15:8693-8711).

Plant gene expression has to be operatively linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al. 1980, Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plant promoterslike those from Rubisco small subunit described in U.S. Pat. No.4,962,028. Even more preferred are seed-specific promoters drivingexpression of LMP proteins during all or selected stages of seeddevelopment. Seed-specific plant promoters are known to those ofordinary skill in the art and are identified and characterized usingseed-specific mRNA libraries and expression profiling techniques.Seed-specific promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al. 1991, Mol. Gen. Genetics 225:459-67), the oleosin-promoter fromArabidopsis (WO 98/45461), the phaseolin-promoter from Phaseolusvulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica(WO9113980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992,Plant J. 2:233-239) as well as promoters conferring seed specificexpression in monocot plants like maize, barley, wheat, rye, rice etc.Suitable promoters to note are the lpt2 or lpt1-gene promoter frombarley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890(promoters from the barley hordein-gene, the rice glutelin gene, therice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheatglutelin gene, the maize zein gene, the oat glutelin gene, the Sorghumkasirin-gene, and the rye secalin gene).

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is desired in a time specific manner. Examples for suchpromoters are a salicylic acid inducible promoter (WO 95/19443), atetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404)and an ethanol inducible promoter (WO 93/21334).

Promoters responding to biotic or abiotic stress conditions are alsosuitable promoters such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993, Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII-promoter (EP 375091).

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene-product in itsappropriate cell compartment (for review see Kermode 1996, Crit. Rev.Plant Sci. 15:285-423 and references cited therein) such as the vacuole,the nucleus, all types of plastids like amyloplasts, chloroplasts,chromoplasts, the extracellular space, mitochondria, the endoplasmicreticulum, oil bodies, peroxisomes and other compartments of plantcells. Also especially suited are promoters that confer plastid-specificgene expression, as plastids are the compartment where precursors andsome end products of lipid biosynthesis are synthesized. Suitablepromoters such as the viral RNA-polymerase promoter are described in WO95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsisdescribed in WO 99/46394.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to LMP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986, AntisenseRNA as a molecular tool for genetic analysis, Reviews—Trends inGenetics, Vol. 1) and Mol et al. (1990, FEBS Lett. 268:427-430).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is to be understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. A host cell can beany prokaryotic or eukaryotic cell. For example, a LMP can be expressedin bacterial cells, insect cells, fungal cells, mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates orplant cells. Other suitable host cells are known to those skilled in theart.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) and other laboratory manuals such asMethods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed:Gartland and Davey, Humana Press, Totowa, N.J.

For stable transfection of mammalian and plant cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin, kanamycin andmethotrexate or in plants that confer resistance towards an herbicidesuch as glyphosate or glufosinate. A nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding a LMP or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by, forexample, drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of a LMP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the LMP gene. Preferably, this LMP gene is anArabidopsis thaliana, Brassica napus, and Physcomitrella patens LMPgene, but it can be a homologue from a related plant or even from amammalian, yeast, or insect source. In a preferred embodiment, thevector is designed such that, upon homologous recombination, theendogenous LMP gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a knock-out vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous LMP gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousLMP). To create a point mutation via homologous recombination, DNA-RNAhybrids can be used in a technique known as chimeraplasty (Cole-Strausset al. 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, AmericanScientist 87:240-247). Homologous recombination procedures inArabidopsis thaliana are also well known in the art and are contemplatedfor use herein.

In a homologous recombination vector, the altered portion of the LMPgene is flanked at its 5′ and 3′ ends by additional nucleic acid of theLMP gene to allow for homologous recombination to occur between theexogenous LMP gene carried by the vector and an endogenous LMP gene in amicroorganism or plant. The additional flanking LMP nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several hundreds of base pairs up tokilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas & Capecchi 1987, Cell 51:503, for adescription of homologous recombination vectors). The vector isintroduced into a microorganism or plant cell (e.g., viapolyethyleneglycol mediated DNA). Cells in which the introduced LMP genehas homologously recombined with the endogenous LMP gene are selectedusing art-known techniques.

In another embodiment, recombinant microorganisms can be produced whichcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a LMP gene on a vectorplacing it under control of the lac operon permits expression of the LMPgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture can be used to produce (i.e., express) a LMP.Accordingly, the invention further provides methods for producing LMPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing a host cell of the invention (into which arecombinant expression vector encoding a LMP has been introduced, orwhich contains a wild-type or altered LMP gene in it's genome) in asuitable medium until LMP is produced. In another embodiment, the methodfurther comprises isolating LMPs from the medium or the host cell.

Another aspect of the invention pertains to isolated LMPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof LMP in which the protein is separated from cellular components of thecells in which it is naturally or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of LMP having less than about 30% (by dry weight)of non-LMP (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-LMP, still more preferably lessthan about 10% of non-LMP, and most preferably less than about 5%non-LMP. When the LMP or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofLMP in which the protein is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of LMP having less than about 30%(by dry weight) of chemical precursors or non-LMP chemicals, morepreferably less than about 20% chemical precursors or non-LMP chemicals,still more preferably less than about 10% chemical precursors or non-LMPchemicals, and most preferably less than about 5% chemical precursors ornon-LMP chemicals. In preferred embodiments, isolated proteins orbiologically active portions thereof lack contaminating proteins fromthe same organism from which the LMP is derived. Typically, suchproteins are produced by recombinant expression of, for example, anArabidopsis thaliana and Brassica napus LMP in other plants thanArabidopsis thaliana and Brassica napus or microorganisms, algae orfungi.

An isolated LMP or a portion thereof of the invention can participate inthe metabolism of compounds necessary for the production of seed storagecompounds in Arabidopsis thaliana and Brassica napus, or of cellularmembranes, or has one or more of the activities set forth in Table 3. Inpreferred embodiments, the protein or portion thereof comprises an aminoacid sequence which is sufficiently homologous to an amino acid sequenceencoded by a nucleic acid of Appendix A such that the protein or portionthereof maintains the ability to participate in the metabolism ofcompounds necessary for the construction of cellular membranes inArabidopsis thaliana and Brassica napus, or in the transport ofmolecules across these membranes. The portion of the protein ispreferably a biologically active portion as described herein. In anotherpreferred embodiment, a LMP of the invention has an amino acid sequenceencoded by a nucleic acid of Appendix A. In yet another preferredembodiment, the LMP has an amino acid sequence which is encoded by anucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, to a nucleotide sequence of Appendix A. In still anotherpreferred embodiment, the LMP has an amino acid sequence which isencoded by a nucleotide sequence that is at least about 50-60%,preferably at least about 60-70%, more preferably at least about 70-80%,80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%,99% or more homologous to one of the amino acid sequences encoded by anucleic acid of Appendix A. The preferred LMPs of the present inventionalso preferably possess at least one of the LMP activities describedherein. For example, a preferred LMP of the present invention includesan amino acid sequence encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, to a nucleotidesequence of Appendix A, and which can participate in the metabolism ofcompounds necessary for the construction of cellular membranes inArabidopsis thaliana and Brassica napus, or in the transport ofmolecules across these membranes, or which has one or more of theactivities set forth in Table 3.

In other embodiments, the LMP is substantially homologous to an aminoacid sequence encoded by a nucleic acid of Appendix A and retains thefunctional activity of the protein of one of the sequences encoded by anucleic acid of Appendix A yet differs in amino acid sequence due tonatural variation or mutagenesis, as described in detail above.Accordingly, in another embodiment, the LMP is a protein which comprisesan amino acid sequence which is at least about 50-60%, preferably atleast about 60-70%, and more preferably at least about 70-80, 80-90,90-95%, and most preferably at least about 96%, 97%, 98%, 99% or morehomologous to an entire amino acid sequence and which has at least oneof the LMP activities described herein. In another embodiment, theinvention pertains to a full Arabidopsis thaliana and Brassica napusprotein which is substantially homologous to an entire amino acidsequence encoded by a nucleic acid of Appendix A.

Dominant negative mutations or trans-dominant suppression can be used toreduce the activity of a LMP in transgenics seeds in order to change thelevels of seed storage compounds. To achieve this a mutation thatabolishes the activity of the LMP is created and the inactivenon-functional LMP gene is overexpressed in the transgenic plant. Theinactive trans-dominant LMP protein competes with the active endogenousLMP protein for substrate or interactions with other proteins anddilutes out the activity of the active LMP. In this way the biologicalactivity of the LMP is reduced without actually modifying the expressionof the endogenous LMP gene. This strategy was used by Pontier et al tomodulate the activity of plant transcription factors (Pontier D, Miao ZH, Lam E, Plant J 2001 September; 27(6):529-38, Trans-dominantsuppression of plant TGA factors reveals their negative and positiveroles in plant defense responses).

Homologues of the LMP can be generated by mutagenesis, e.g., discretepoint mutation or truncation of the LMP. As used herein, the term“homologue” refers to a variant form of the LMP which acts as an agonistor antagonist of the activity of the LMP. An agonist of the LMP canretain substantially the same, or a subset, of the biological activitiesof the LMP. An antagonist of the LMP can inhibit one or more of theactivities of the naturally occurring form of the LMP, by, for example,competitively binding to a downstream or upstream member of the cellmembrane component metabolic cascade which includes the LMP, or bybinding to a LMP which mediates transport of compounds across suchmembranes, thereby preventing translocation from taking place.

In an alternative embodiment, homologues of the LMP can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the LMP for LMP agonist or antagonist activity. In one embodiment, avariegated library of LMP variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of LMP variants can be produced by,for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential LMP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of LMP sequences therein. There are avariety of methods which can be used to produce libraries of potentialLMP homologues from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential LMP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang 1983,Tetrahedron 39:3; Itakura et al. 1984, Armu Rev. Biochem. 53:323;Itakura et al. 1984, Science 198:1056; Ike et al. 1983, Nucleic AcidsRes. 11:477).

In addition, libraries of fragments of the LMP coding sequences can beused to generate a variegated population of LMP fragments for screeningand subsequent selection of homologues of a LMP. In one embodiment, alibrary of coding sequence fragments can be generated by treating adouble stranded PCR fragment of a LMP coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the LMP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of LMP homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify LMP homologues (Arkin & Yourvan 1992, Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. 1993, Protein Engineering 6:327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated LMP library, using methods well known in the art.

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification of Arabidopsisthaliana and Brassica napus and related organisms; mapping of genomes oforganisms related to Arabidopsis thaliana and Brassica napus;identification and localization of Arabidopsis thaliana and Brassicanapus sequences of interest; evolutionary studies; determination of LMPregions required for function; modulation of a LMP activity; modulationof the metabolism of one or more cell functions; modulation of thetransmembrane transport of one or more compounds; and modulation of seedstorage compound accumulation.

The plant Arabidopsis thaliana represents one member of higher (or seed)plants. It is related to other plants such as Brassica napus or soybeanwhich require light to drive photosynthesis and growth. Plants likeArabidopsis thaliana and Brassica napus share a high degree of homologyon the DNA sequence and polypeptide level, allowing the use ofheterologous screening of DNA molecules with probes evolving from otherplants or organisms, thus enabling the derivation of a consensussequence suitable for heterologous screening or functional annotationand prediction of gene functions in third species. The ability toidentify such functions can therefore have significant relevance, e.g.,prediction of substrate specificity of enzymes. Further, these nucleicacid molecules may serve as reference points for the mapping ofArabidopsis genomes, or of genomes of related organisms.

The LMP nucleic acid molecules of the invention have a variety of uses.First, they may be used to identify an organism as being Arabidopsisthaliana, Brassica napus, and Physcomitrella patens or a close relativethereof. Also, they may be used to identify the presence of Arabidopsisthaliana, Brassica napus, and Physcomitrella patens or a relativethereof in a mixed population of microorganisms. The invention providesthe nucleic acid sequences of a number of Arabidopsis thaliana andBrassica napus genes; by probing the extracted genomic DNA of a cultureof a unique or mixed population of microorganisms under stringentconditions with a probe spanning a region of an Arabidopsis thaliana andBrassica napus gene which is unique to this organism, one can ascertainwhether this organism is present.

Further, the nucleic acid and protein molecules of the invention mayserve as markers for specific regions of the genome. This has utilitynot only in the mapping of the genome, but also for functional studiesof Arabidopsis thaliana and Brassica napus proteins. For example, toidentify the region of the genome to which a particular Arabidopsisthaliana and Brassica napus DNA-binding protein binds, the Arabidopsisthaliana and Brassica napus genome could be digested, and the fragmentsincubated with the DNA-binding protein. Those which bind the protein maybe additionally probed with the nucleic acid molecules of the invention,preferably with readily detectable labels; binding of such a nucleicacid molecule to the genome fragment enables the localization of thefragment to the genome map of Arabidopsis thaliana and Brassica napus,and, when performed multiple times with different enzymes, facilitates arapid determination of the nucleic acid sequence to which the proteinbinds. Further, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species such thatthese nucleic acid molecules may serve as markers for the constructionof a genomic map in related plants.

The LMP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein which are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the LMP nucleic acid molecules of the invention mayresult in the production of LMPs having functional differences from thewild-type LMPs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a LMP of theinvention may directly affect the accumulation of seed storagecompounds. In the case of plants expressing LMPs, increased transportcan lead to altered accumulation of compounds and/or solute partitioningwithin the plant tissue and organs which ultimately could be used toaffect the accumulation of one or more seed storage compounds duringseed development. An example is provided by Mitsukawa et al. (1997,Proc. Natl. Acad. Sci. USA 94:7098-7102), where over expression of anArabidopsis high-affinity phosphate transporter gene in tobacco culturedcells enhanced cell growth under phosphate-limited conditions. Phosphateavailability also affects significantly the production of sugars andmetabolic intermediates (Hurry et al. 2000, Plant J. 24:383-396) and thelipid composition in leaves and roots (Härtel et al. 2000, Proc. Natl.Acad. Sci. USA 97:10649-10654). Likewise, the activity of the plantACCase has been demonstrated to be regulated by phosphorylation (Savage& Ohlrogge 1999, Plant J. 18:521-527) and alterations in the activity ofthe kinases and phosphatases (LMPs) that act on the ACCase could lead toincreased or decreased levels of seed lipid accumulation. Moreover, thepresence of lipid kinase activities in chloroplast envelope membranessuggests that signal transduction pathways and/or membrane proteinregulation occur in envelopes (see, e.g., Müller et al. 2000, J. Biol.Chem. 275:19475-19481 and literature cited therein). The ABI1 and ABI2genes encode two protein serine/threonine phosphatases 2C, which areregulators in abscisic acid signaling pathway, and thereby in early andlate seed development (e.g. Merlot et al. 2001, Plant J. 25:295-303).For more examples see also the section ‘background of the invention’.

The present invention also provides antibodies which specifically bindsto an LMP-polypeptide, or a portion thereof, as encoded by a nucleicacid disclosed herein or as described herein.

Antibodies can be made by many well-known methods (see, e.g. Harlow andLane, “Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988). Briefly, purified antigen can beinjected into an animal in an amount and in intervals sufficient toelicit an immune response. Antibodies can either be purified directly,or spleen cells can be obtained from the animal. The cells can thenfused with an immortal cell line and screened for antibody secretion.The antibodies can be used to screen nucleic acid clone libraries forcells secreting the antigen. Those positive clones can then be sequenced(see, for example, Kelly et al. 1992, Bio/Technology 10:163-167;Bebbington et al. 1992, Bio/Technology 10:169-175).

The phrase “selectively binds” with the polypeptide refers to a bindingreaction which is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bound to aparticular protein do not bind in a significant amount to other proteinspresent in the sample. Selective binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. A variety of immunoassay formats may be usedto select antibodies that selectively bind with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein. See Harlow andLane “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York (1988), for a description of immunoassay formats and conditionsthat could be used to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane(“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, NewYork, 1988).

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andExamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the claims included herein.

EXAMPLES Example 1 General Processes a) General Cloning Processes:

Cloning processes such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli and yeast cells, growth of bacteriaand sequence analysis of recombinant DNA were carried out as describedin Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994, “Methods inYeast Genetics,” Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3).

b) Chemicals:

The chemicals used were obtained, if not mentioned otherwise in thetext, in p.a. quality from the companies Fluka (Neu-Ulm), Merck(Darmstadt), Roth (Karlsruhe), Serva (Heidelberg), and Sigma(Deisenhofen). Solutions were prepared using purified, pyrogen-freewater, designated as H₂O in the following text, from a Milli-Q watersystem water purification plant (Millipore, Eschborn). Restrictionendonucleases, DNA-modifying enzymes, and molecular biology kits wereobtained from the companies AGS (Heidelberg), Amersham (Braunschweig),Biometra (Göttingen), Boehringer (Mannheim), Genomed (Bad Oeynnhausen),New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wis., USA),Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden), andStratagene (Amsterdam, Netherlands). They were used, if not mentionedotherwise, according to the manufacturer's instructions.

c) Plant Material: Arabidopsis pkl Mutant

For this study, in one series of experiments, root material of wild-typeand pickle mutant Arabidopsis thaliana plants were used. The pklmutation was isolated from an ethyl methanesulfonate-mutagenizedpopulation of the Columbia ecotype as described (Ogas et al., 1997,Science 277:91-94; Ogas et al., 1999, Proc. Natl. Acad. Sci. USA96:13839-13844). In other series of experiments, siliques of individualecotypes of Arabidopsis thaliana and of selected Arabidopsisphytohormone mutants were used. Seeds were obtained from the Arabidopsisstock center.

Brassica napus AC Excel and Cresor Varieties

Brassica napus varieties AC Excel and Cresor were used for this study tocreate cDNA libraries. Seed, seed pod, flower, leaf, stem, and roottissues were collected from plants that were in some cases dark-, salt-,heat-, and drought-treated. However, this study focused on the use ofseed and seed pod tissues for cDNA libraries.

d) Plant Growth:

Arabidopsis thaliana

Plants were either grown on Murashige-Skoog medium as described in Ogaset al. (1997, Science 277:91-94; 1999, Proc. Natl. Acad. Sci. USA96:13839-13844) or on soil under standard conditions as described inFocks & Benning (1998, Plant Physiol. 118:91-101).

Brassica napus

Plants (AC Excel, except where mentioned) were grown in Metromix(Scotts, Marysville, Ohio) at 22° C. under a 14/10 light/dark cycle. Sixseed and seed pod tissues of interest in this study were collected tocreate the following cDNA libraries: Immature seeds, mature seeds,immature seed pods, mature seed pods, night-harvested seed pods, andCresor variety (high erucic acid) seeds. Tissue samples were collectedwithin specified time points for each developing tissue and multiplesamples within a time frame pooled together for eventual extraction oftotal RNA. Samples from immature seeds were taken between 1-25 daysafter anthesis (daa), mature seeds between 25-50 daa, immature seed podsbetween 1-15 daa, mature seed pods between 15-50 daa, night-harvestedseed pods between 1-50 daa and Cresor seeds 5-25 daa.

Example 2 Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material.

CTAB buffer: 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB);100 mM Tris HCl pH 8.0; 1.4 M NaCl; 20 mM EDTA. N-Laurylsarcosinebuffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000 gand RT for 15 minutes in each case. The DNA was then precipitated at−70° C. for 30 minutes using ice-cold isopropanol. The precipitated DNAwas sedimented at 4° C. and 10,000 g for 30 minutes and resuspended in180 μl of TE buffer (Sambrook et al., 1989, Cold Spring HarborLaboratory Press: ISBN 0-87969-309-6). For further purification, the DNAwas treated with NaCl (1.2 M final concentration) and precipitated againat −70° C. for 30 minutes using twice the volume of absolute ethanol.After a washing step with 70% ethanol, the DNA was dried andsubsequently taken up in 50 μl of H2O+RNAse (50 mg/ml finalconcentration). The DNA was dissolved overnight at 4° C. and the RNAsedigestion was subsequently carried out at 37° C. for 1 hour. Storage ofthe DNA took place at 4° C.

Example 3 Isolation of Total RNA and Poly-(A)+ RNA from Plants

Arabidopsis thaliana

For the investigation of transcripts, both total RNA and poly-(A)+ RNAwere isolated. RNA was isolated from siliques of Arabidopsis plantsaccording to the following procedure:

RNA preparation from Arabidopsis seeds—“hot” extraction:

Buffers, Enzymes, and Solutions:

2M KCl

Proteinase K

Phenol (for RNA)

Chloroform:Isoamylalcohol

(Phenol:choloroform 1:1; pH adjusted for RNA)

4 M LiCl, DEPC-treated

DEPC-treated water

3M NaOAc, pH 5, DEPC-treated

Isopropanol

70% ethanol (made up with DEPC-treated water)

Resuspension buffer: 0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up withDEPC-treated water as this solution can not be DEPC-treated

Extraction Buffer:

0.2M Na Borate

30 mM EDTA

30 mM EGTA

1% SDS (250 μl of 10% SDS-solution for 2.5 ml buffer)

-   -   1% Deoxycholate (25 mg for 2.5 ml buffer)

2% PVPP (insoluble—50 mg for 2.5 ml buffer)

2% PVP 40K (50 mg for 2.5 ml buffer)

10 mM DTT

100 mM β-Mercaptoethanol (fresh, handle under fume hood—use 35 μl of14.3M solution for 5 ml buffer)

Extraction

Extraction buffer was heated up to 80° C. Tissues were ground in liquidnitrogen-cooled mortar, and the tissue powder was transferred to a 1.5ml tube. Tissues should be kept frozen until buffer is added; the sampleshould be transferred with a pre-cooled spatula; and the tube should bekept in liquid nitrogen at all times. Then 350 μl preheated extractionbuffer was added (For 100 mg tissue, buffer volume can be as much as 500μl for bigger samples) to tube; samples were vortexed; and the tube washeated to 80° C. for approximately 1 minute and then kept on ice. Thesamples were vortexed and ground additionally with electric mortar.

Digestion

Proteinase K (0.15 mg/100 mg tissue) was added, and the mixture wasvortexed and then kept at 37° C. for one hour.

First Purification

For purification, 27 μl 2M KCl was added to the samples. The sampleswere chilled on ice for 10 minutes and then centrifuged at 12.000 rpmfor 10 minutes at room temperature. The supernatant was transferred to afresh, RNAase-free tube, and one phenol extraction was conducted,followed by a choloroform:isoamylalcohol extraction. One volumeisopropanol to was added to the supernatant, and the mixture was chilledon ice for 10 minutes. RNA was pelleted by centrifugation (7000 rpm for10 minutes at room temperature). Pellets were dissolved in 1 ml 4M LiClsolution by vortexing the mixture 10 to 15 minutes. RNA was pelleted bya 5 minute centrifugation.

Second Purification

The pellet was resuspended in 500 μl Resuspension buffer. Then 500 μl ofphenol was added, and the mixture was vortexed. Then, 250 μlchloroform:isoamylalcohol was added; the mixture was vortexed and thencentrifuged for 5 minutes. The supernatant was transferred to a freshtube. The choloform:isoamylalcohol extraction was repeated until theinterface was clear. The supernatant was transferred to a fresh tube and1/10 volume 3M NaOAc, pH 5 and 600 μl isopropanol were added. Themixture was kept at −20 for 20 minutes or longer. The RNA was pelletedby 10 minutes of centrifugation, and then the pellet was washed oncewith 70% ethanol. All remaining alcohol was removed before dissolvingthe pellet in 15 to 20 μl DEPC-treated water. The quantity and qualityof the RNA was determined by measuring the absorbance of a 1:200dilution at 260 nm and 280 nm. (40 μg RNA/ml=1 OD₂₆₀)

RNA from roots of wild-type Arabidopsis and the pickle mutant ofArabidopsis was isolated as described (Ogas et al., 1997, Science277:91-94; Ogas et al., 1999, Proc. Natl. Acad. Sci. USA96:13839-13844).

The mRNA was prepared from total RNA, using the Amersham PharmaciaBiotech mRNA purification kit, which utilizes oligo(dT)-cellulosecolumns.

Isolation of Poly-(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo,Norway) following the instructions of the manufacturer's protocol. Afterdetermination of the concentration of the RNA or of the poly(A)+ RNA,the RNA was precipitated by addition of 1/10 volume of 3 M sodiumacetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

Brassica napus

Seeds were separated from pods to create homogeneous materials for seedand seed pod cDNA libraries. Tissues were ground into fine powder underliquid nitrogen using a mortar and pestle and transferred to a 50 mltube. Tissue samples were stored at −80° C. until extractions could beperformed. Total RNA was extracted from tissues using RNeasy Maxi kit(Qiagen) according to manufacturer's protocol, and mRNA was processedfrom total RNA using Oligotex mRNA Purification System kit (Qiagen),also according to manufacturer's protocol. The mRNA was sent to HyseqPharmaceuticals Incorporated (Sunnyville, Calif.) for further processingof mRNA from each tissue type into cDNA libraries and for use in theirproprietary processes in which similar inserts in plasmids are clusteredbased on hybridization patterns.

Example 4 cDNA Library Construction

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche,Mannheim) at 37° C. (30 minutes). Nucleotides were removed byphenol/chloroform extraction and Sephadex G50 spin columns EcoRIadapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends byT4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated byincubation with polynucleotide kinase (Roche, 37° C., 30 minutes). Thismixture was subjected to separation on a low melting agarose gel. DNAmolecules larger than 300 base pairs were eluted from the gel, phenolextracted, concentrated on Elutip-D-columns (Schleicher and Schuell,Dassel, Germany) and were ligated to vector arms and packed into lambdaZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit(Stratagene, Amsterdam, Netherlands) using material and following theinstructions of the manufacturer.

Brassica cDNA libraries were generated at Hyseq PharmaceuticalsIncorporated (Sunnyville, Calif.) No amplification steps were used inthe library production to retain expression information. Hyseq's genomicapproach involves grouping the genes into clusters and then sequencingrepresentative members from each cluster. The cDNA libraries weregenerated from oligo dT column purified mRNA. Colonies fromtransformation of the cDNA library into E. coli were randomly picked andthe cDNA insert were amplified by PCR and spotted on nylon membranes. Aset of ³³⁻P radiolabeled oligonucleotides were hybridized to the clones,and the resulting hybridization pattern determined to which cluster aparticular clone belonged. The cDNA clones and their DNA sequences wereobtained for use in overexpression in transgenic plants and in othermolecular biology processes described herein.

Example 5 Identification of LMP Genes of Interest

Arabidopsis thaliana pkl Mutant

The pickle Arabidopsis mutant was used to identify LMP-encoding genes.The pickle mutant accumulates seed storage compounds, such as seedstorage lipids and seed storage proteins, in the root tips (Ogas et al.,1997, Science 277:91-94; Ogas et al., 1999, Proc. Natl. Acad. Sci. USA96:13839-13844). The mRNA isolated from roots of wild-type and pickleplants was used to create a subtracted and normalized cDNA library (SSHlibrary) containing cDNAs that are only present in the pickle roots, butnot in the wild-type roots. Clones from the SSH library were spottedonto nylon membranes and hybridized with radio-labeled pickle orwild-type root mRNA to ascertain that the SSH clones were more abundantin pickle roots compared to wild-type roots. These SSH clones wererandomly sequenced and the sequences were annotated (See Example 9).Based on the expression levels and on these initial functionalannotations (See Table 3), clones from the SSH library were identifiedas potential LMP-encoding genes.

To identify additional potential gene targets from the Arabidopsispickle mutant, the Megasort™ and MPSS technologies of Lynx TherapeuticsInc. were used. MegaSort is a micro-bead technology that allows both thesimultaneous collection of millions of clones on as many micro-beads(See Brenner et al., 1999, Proc. Natl. Acad. Sci. USA 97:1665-1670).Genes are identified based on their differential expression in wild-typeand pickle Arabidopsis mutant roots. RNA and mRNA are isolated fromwild-type and mutant roots using standard procedures. The MegaSorttechnology enables the identification of over- and under-expressedclones in two mRNA samples without prior knowledge of the genes and isthus useful to discover differentially expressed genes that can encodeLMP proteins. The MPSS technology enables the quantitation of theabundance of mRNA transcripts in mRNA samples (Brenner et al., Nat.Biotechnol. 18:630-4) and was used to obtain expression profiles ofwild-type and pickle root mRNAs.

Other LMP candidate genes were identified by randomly selecting variousArabidopsis phytohormone mutants (e.g. mutants obtained from EMStreatment) from the Arabidopsis stock center. These mutants and controlwild-type plants were grown under standard conditions in growth chambersand screened for the accumulation of seed storage compounds. Mutantsshowing altered levels of seed storage compounds were considered ashaving a mutation in a LMP candidate gene and were investigated further.

Brassica napus

RNA expression profile data was obtained from the Hyseq clusteringprocess. Clones showing 75% or greater expression in seed librariescompared to the other tissue libraries were selected as LMP candidategenes. The Brassica napus clones were selected for overexpression inArabidopsis based on their expression profile.

Example 6 Cloning of Full-Length cDNAs and Orthologs of Identified LMPGenes

Arabidopsis thaliana

Full-length sequences of the Arabidopsis thaliana partial cDNAs (ESTs)that were identified in the SSH library and from MegaSort and MPSS ESTsequencing were isolated by RACE PCR using the SMART RACE cDNAamplification kit from Clontech allowing both 5′ and 3′ rapidamplification of cDNA ends (RACE). The isolation of cDNAs and the RACEPCR protocol used were based on the manufacturer's conditions. The RACEproduct fragments were extracted from agarose gels with a QIAquick GelExtraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector(Invitrogen) following manufacturer's instructions. Recombinant vectorswere transformed into TOP10 cells (Invitrogen) using standard conditions(Sambrook et al., 1989). Transformed cells were grown overnight at 37°C. on LB agar containing 50 μg/ml kanamycin and spread with 40 μl of a40 mg/ml stock solution of X-gal in dimethylformamide for blue-whiteselection. Single white colonies were selected and used to inoculate 3ml of liquid LB containing 50 μg/ml kanamycin and grown overnight at 37°C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit(Qiagen) following manufacturer's instructions. Subsequent analyses ofclones and restriction mapping was performed according to standardmolecular biology techniques (Sambrook et al., 1989).

Gene sequences can be used to identify homologous or heterologous genes(orthologs, the same LMP gene from another plant) from cDNA or genomiclibraries. This can be done by designing PCR primers to conservedsequences identified by multiple sequence alignments. Orthologs areoften identified by designing degenerate primers to full-length orpartial sequences of genes of interest. Homologous genes (e.g.full-length cDNA clones) can be isolated via nucleic acid hybridizationusing, for example, cDNA libraries: Depending on the abundance of thegene of interest, 100,000 up to 1,000,000 recombinant bacteriophages areplated and transferred to nylon membranes. After denaturation withalkali, DNA is immobilized on the membrane by e.g. UV cross linking.Hybridization is carried out at high stringency conditions. Aqueoussolution hybridization and washing is performed at an ionic strength of1 M NaCl and a temperature of 68° C. Hybridization probes are generatedby, e.g., radioactive (³²P) nick transcription labeling (High Prime,Roche, Mannheim, Germany). Signals are detected by autoradiography.

Partially homologous or heterologous genes that are related but notidentical can be identified in a procedure analogous to theabove-described procedure using low stringency hybridization and washingconditions. For aqueous hybridization, the ionic strength is normallykept at 1 M NaCl while the temperature is progressively lowered from 68to 42° C.

Isolation of gene sequences with homology (or sequenceidentity/similarity) only in a distinct domain (for example 10-20 aminoacids) can be carried out by using synthetic radiolabeledoligonucleotide probes. Radiolabeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

Oligonucleotide Hybridization Solution: 6×SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8) 0.5% SDS

100 μg/ml denaturated salmon sperm DNA0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide T_(m) or down to room temperature followedby washing steps and autoradiography. Washing is performed with lowstringency such as three washing steps using 4×SSC. Further details aredescribed by Sambrook et al. (1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press) or Ausubel et al. (1994,“Current Protocols in Molecular Biology”, John Wiley & Sons).

Brassica napus

Clones of Brassica napus genes obtained from Hyseq were sequenced atusing a ABI 377 slab gel sequencer and BigDye Terminator Ready Reactionkits (PE Biosystems, Foster City, Calif.). Gene specific primers weredesigned using these sequences, and genes were amplified from theplasmid supplied from Hyseq using touch-down PCR. In some cases, primerswere designed to add an “AACA” Kozak-like sequence just upstream of thegene start codon and two bases downstream were, in some cases, changedto GC to facilitate increased gene expression levels (Chandrashekhar etal., 1997, Plant Molecular Biology 35:993-1001). PCR reaction cycleswere: 94° C., 5 minutes; 9 cycles of 94° C., 1 minute, 65° C., 1 minute,72° C., 4 minutes and in which the anneal temperature was lowered by 1°C. each cycle; 20 cycles of 94° C., 1 minute, 55° C., 1 minute, 72° C.,4 minutes; and the PCR cycle was ended with 72° C., 10 minutes.Amplified PCR products were gel purified from 1% agarose gels usingGenElute-EtBr spin columns (Sigma), and after standard enzymaticdigestion, were ligated into the plant binary vector pBPS-GB1 fortransformation of Arabidopsis. The binary vector was amplified byovernight growth in E. coli DH5 in LB media and appropriate antibiotic,and plasmid was prepared for downstream steps using Qiagen MiniPrep DNApreparation kit. The insert was verified throughout the various cloningsteps by determining its size through restriction digest and insertswere sequenced in parallel to plant transformations to ensure theexpected gene was used in Arabidopsis transformation.

RT-PCR and Cloning of Arabidopsis thaliana, Brassica napus, andPhyscomitrella patens LMP Genes

Full-length LMP cDNAs were isolated by RT-PCR from Arabidopsis thaliana,Brassica napus, or Physcomitrella patens RNA. The synthesis of the firststrand cDNA was achieved using AMV Reverse Transcriptase (Roche,Mannheim, Germany). The resulting single-stranded cDNA was amplified viaPolymerase Chain Reaction (PCR) utilizing two gene-specific primers. Theconditions for the reaction were standard conditions with Expand HighFidelity PCR system (Roche). The parameters for the reaction were: fiveminutes at 94° C. followed by five cycles of 40 seconds at 94° C., 40seconds at 50° C., and 1.5 minutes at 72° C. This was followed by thirtycycles of 40 seconds at 94° C., 40 seconds at 65° C., and 1.5 minutes at72° C. The fragments generated under these RT-PCR conditions wereanalyzed by agarose gel electrophoresis to make sure that PCR productsof the expected length had been obtained.

Full-length LMP cDNAs were isolated by using synthetic oligonucleotideprimers (MWG-Biotech) designed based on the LMP gene specific DNAsequence that was determined by EST sequencing and by sequencing of RACEPCR products. The 5′ PCR primers (“forward primer”, F) for SEQ ID NO:83,SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ IDNO:113, and SEQ ID NO:115 contained an AscI restriction site 5′ upstreamof the ATG start codon. The 5′ PCR primers (“forward primer”, F) for SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:135, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:49, and SEQ ID NO:131, containeda NotI restriction site 5′ upstream of the ATG start codon. The 3′ PCRprimers (“reverse primers”, R) for SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, and SEQ IDNO:116 contained a PacI restriction site 3′ downstream of the stopcodon. The 3′ PCR primers (“reverse primers”, R) for SEQ ID NO:118, SEQID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128,SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, and SEQ IDNO:140, contained a NotI restriction site 3′ downstream of the stopcodon. The 3′ PCR primers (“reverse primers”, R) for SEQ ID NO:142, SEQID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:50, and SEQ IDNO:132, contained a StuI restriction site 3′ downstream of the stopcodon. The 3′ PCR primers (“reverse primers”, R) for SEQ ID NO:154contained an EcoRV restriction site 3′ downstream of the stop codon.

The restriction sites were added so that the RT-PCR amplificationproducts could be cloned into the restriction sites located in themultiple cloning site of the binary vector. The following “forward” (F)and “reverse” (R) primers were used to amplify the full-lengthArabidopsis thaliana or Brassica napus cDNAs by RT-PCR using RNA fromArabidopsis thaliana or Brassica napus as original template:

For amplification of SEQ ID NO: 1 Pk123F (SEQ ID NO: 83)(5′-ATGGCGCGCCATGGCAATCTTCCGAAGTACACTAGT-3′) Pk123R (SEQ ID NO: 84)(5′-GCTTAATTAATTAAGGGCACTTGAGACGGCCA-3′) For amplification of SEQ ID NO:3 Pk197F (SEQ ID NO: 85) (5′-ATGGCGCGCCAACAATGGAGAATGGAGCAACGACG-3′)Pk197R (SEQ ID NO: 86) (5′-GCTTAATTAACTATATGGTTGGATATTGAGTCTTGGC-3′) Foramplification of SEQ ID NO: 5 Pk136F (SEQ ID NO: 87)(5′-ATGGCGCGCCATGGCTGAAAAAGTAAAGTCTGGTCA-3′) Pk136R (SEQ ID NO: 88)(5′-GCTTAATTAATTATAGCTCCTCAGATCCCTCCGA-3′) For amplification of SEQ IDNO: 7 Pk156F (SEQ ID NO: 89) (5′-ATGGCGCGCCATGGCTGGAGAAGAAATAGAGAGGG-3′)Pk156R (SEQ ID NO: 90) (5′-GCTTAATTAATTAAACAGAGGCTTCTCTACTCTCACTT-3′)For amplification of SEQ ID NO: 9 Pk159F (SEQ ID NO: 91)(5′-ATGGCGCGCCATGGCTGGAGTGATGAAGTTGGC-3′) Pk159R (SEQ ID NO: 92)(5′-GCTTAATTAATCACCTCACGGTGTTGCAGTTG-3′) For amplification of SEQ ID NO:11 Pk179F (SEQ ID NO: 93) (5′-ATGGCGCGCCAAACAATGGGGCTTGCTGTGGTGG-3′)Pk179R (SEQ ID NO: 94) (5′-GCTTAATTAATTACTGCAAGGCTTTCAATATATTTC-3′) Foramplification of SEQ ID NO: 13 Pk202F (SEQ ID NO: 95)(5′-ATGGCGCGCCAACAATGGCGTTCACGGCGCTTGT-3′) Pk202R (SEQ ID NO: 96)(5′-GCTTAATTAATCAACAAGTAGGATAAGGAACACCACA-3′) For amplification of SEQID NO: 15 Pk206F (SEQ ID NO: 97)(5′-ATGGCGCGCCAACAATGGCCCTTGATGAGCTTCTCAAG-3′) Pk206R (SEQ ID NO: 98)(5′-GCTTAATTAATCAGAGAGAAGCAGAGTTTGTTCGC-3′) For amplification of SEQ IDNO: 17 Pk207F (SEQ ID NO: 99)(5′-ATGGCGCGCCAACAATGGCGCAATCCCGATTATTAG-3′) Pk207R (SEQ ID NO: 100)(5′-GCTTAATTAATTAAAACCACTCGCCTCTCATTTC-3′) For amplification of SEQ IDNO: 19 Pk209F (SEQ ID NO: 101) (5′-ATGGCGCGCCATGTCCGTGGCTCGATTCGAT-3′)Pk209R (SEQ ID NO: 102) (5′-GCTTAATTAACTAATCCTCTAGCTCGATGATTTTGAC-3′)For amplification of SEQ ID NO: 21 Pk215F (SEQ ID NO: 103)(5′-ATGGCGCGCCAACAATGGCGATTTACAGATC TCTAAGAAAG-3′) Pk215R (SEQ ID NO:104) (5′-GCTTAATTAATTACCTTAGATAAGTGATCCATGTCTGG-3′) For amplification ofSEQ ID NO: 23 Pk239F (SEQ ID NO: 105) (5′-ATGGCGCGCCAACAATGGTAAAGGAAACTCTAATTCCTCCG-3′) Pk239R (SEQ ID NO: 106)(5′-GCTTAATTAACTACCAGCCGAAGATTGGCTTGT-3′) For amplification of SEQ IDNO: 25 Pk240F (SEQ ID NO: 107) (5′-ATGGCGCGCCATTTGGAGAGCAATGGCGACTT-3′)Pk240R (SEQ ID NO: 108) (5′-GCTTAATTAATTACATCGAACGAAGAAGC ATCAA-3′) Foramplification of SEQ ID NO: 27 Pk241F (SEQ ID NO: 109)(5′-ATGGCGCGCCCATCCTCAGAAAGAATGGCTCAAA-3′) Pk241R (SEQ ID NO: 110)(5′-GCTTAATTAATTAGCTTTCTTCACCATCATC GGTG-3′) For amplification of SEQ IDNO: 29 Pk242F (SEQ ID NO: 111)(5′-ATGGCGCGCCAACAATGGGTGCAGGTGGAAGAATGCC-3′) Pk242R (SEQ ID NO: 112)(5′-GCTTAATTAATCATAACTTATTGTTGTACCAGTA CACACC-3′) For amplification ofSEQ ID NO: 31 Bn011F (SEQ ID NO: 113) (5′-ATGGCGCGCCAACAATGGCTTCAATAAATGAAGATGTGTCT-3′) Bn011R (SEQ ID NO: 114)(5′-GACTTAATTAATCAATTGGTGGGATTAACGA CTCCA-3′) For amplification of SEQID NO: 33 Bn077F (SEQ ID NO: 115) (5′-ATGGCGCGCCAACAATGGCTACATTCTCTTGTAATTCTTATGA-3′) Bn077R (SEQ ID NO: 116)(5′-GACTTAATTAATCAGAAGCGGCCATTAAAATT ACCCA-3′) For amplification of SEQID NO: 35 Jb001F (SEQ ID NO: 117)(5′-ATAAGAATGCGGCCGCCATGGCAACGGAATGCATTGCA-3′) Jb001R (SEQ ID NO: 118)(5′-ATAAGAATGCGGCCGCTTAGAAACTTCT TCTGTTCTT-3′) For amplification of SEQID NO: 37 Jb002F (SEQ ID NO: 119) (5′-ATAAGAATGCGGCCGCCATGGCGTCAGAGCAAGCAAGG-3′) Jb002R (SEQ ID NO: 120) (5′-ATAAGAATGCGGCCGCTCAACGTTGTCCATGTTCCCG-3′) For amplification of SEQ ID NO: 39 Jb003F (SEQ ID NO: 121)(5′-ATAAGAATGCGGCCGCCATGGCTAAGTC TTGCTATTTCA-3′) Jb003R (SEQ ID NO: 122)(5′-ATAAGAATGCGGCCGCTCAGGCGCTATAG CCTAAGATT-3′) For amplification of SEQID NO: 41 Jb005F (SEQ ID NO: 123) (5′-ATAAGAATGCGGCCGCCATGGACGGTGCCGGAGAATCACGA-3′) Jb005R (SEQ ID NO: 124) (5′-ATAAGAATGCGGCCGCCTAATAACTTAAAGTTACCGGA-3′) For amplification of SEQ ID NO: 43 Jb007F (SEQ ID NO:125) (5′-ATAAGAATGCGGCCGCCATGTCGAGAGCTTTG TCAGTCG-3′) Jb007R (SEQ ID NO:126) (5′-ATAAGAATGCGGCCGCCATGTCGAGAGCTTT GTCAGTCG-3′) For amplificationof SEQ ID NO: 45 Jb009F (SEQ ID NO: 127)(5′-ATAAGAATGCGGCCGCCATGGCAAGCAGCGAC GTGAAGCT-3′) Jb009R (SEQ ID NO:128) (5′-ATAAGAATGCGGCCGCTCAACCAAGCCAAGAA GCACCC-3′) For amplificationof SEQ ID NO: 47 Jb013F (SEQ ID NO: 129)(5′-ATAAGAATGCGGCCGCCATGGCGTCTCAACAAGA GAAGA-3′) Jb013R (SEQ ID NO: 130)(5′-ATAAGAATGCGGCCGCTTAGGTCTTGGTCCTGA ATTTG-3′) For amplification of SEQID NO: 51 Jb017F (SEQ ID NO: 133) (5′-ATAAGAATGCGGCCGCCATGGCTCCTTCAACAAAAGTTC-3′) Jb017R (SEQ ID NO: 134)(5′-ATAAGAATGCGGCCGCTCAAACACTGCTGATAGTATTT-3′) For amplification of SEQID NO: 53 Jb024F (SEQ ID NO: 135) (5′-ATAAGAATGCGGCCGCCATGCGGTGCTTTCCACCTCCCT-3′) Jb024R (SEQ ID NO: 136)(5′-ATAAGAATGCGGCCGCTTACTTTTGTAATGGTGAG AGC-3′) For amplification of SEQID NO: 55 Jb027F (SEQ ID NO: 137) (5′-ATAAGAATGCGGCCGCCATGCTTCTAATTCTAGCGATTT-3′) Jb027R (SEQ ID NO: 138)(5′-ATAAGAATGCGGCCGCTCAGATAACCTTCTTCTTCTCG-3′) For amplification of SEQID NO: 57 OO-1F (SEQ ID NO: 139)(5′-ATTGCGGCCGCACAATGGCACATGCCACGTTTACG-3′) OO-1R (SEQ ID NO: 140)(5′-ATTGCGGCCGCTTAGTCTTCATGGTCCCATAGATC-3′) For amplification of SEQ IDNO: 59 OO-2F (SEQ ID NO: 141) (5′-GCGGCCGCCATGGCGTCTGAGAAACAAAAAC-3′)OO-2R (SEQ ID NO: 142) (5′-AGGCCTTTACGCATTTACCACAGCTCC-3′) Foramplification of SEQ ID NO: 61 OO-3F (SEQ ID NO: 143)(5′-GCGGCCGCATGGATTCAACGAAGCTTAGTGAGC-3′) OO-3R (SEQ ID NO: 144)(5′-AGGCCTTTACTGAGGTCCTGCAAATTTG-3′) For amplification of SEQ ID NO: 63OO-4F (SEQ ID NO: 145) (5′-GCGGCCGCCATGAAGGTTCACGAGACAAGA-3′) OO-4R (SEQID NO: 146) (5′-AGGCCTCTACTCTGGTTCGACATCGAC-3′) For amplification of SEQID NO: 65 OO-5F (SEQ ID NO: 147) (5′-GCGGCCGCCATGTCTACCCCAGCTGAATC-3′)OO-5R (SEQ ID NO: 148) (5′-AGGCCTCTAATTGTAGAGATCATCATC-3′) Foramplification of SEQ ID NO: 67 OO-6F (SEQ ID NO: 149)(5′-GCGGCCGCCATGGACAAATCTAGTACCATG-3′) OO-6R (SEQ ID NO: 150)(5′-AGGCCTTCAGCTACCACCCTTTTGTTTGAG-3′) For amplification of SEQ ID NO:69 OO-8F (SEQ ID NO: 151) (5′-GCGGCCGCCATGGCGAAATCTCAGATCTGG-3′) OO-8R(SEQ ID NO: 152) (5′-AGGCCTTTAAGAAGAAGCAACGAACGTG-3′) For amplificationof SEQ ID NO: 71 OO-9F (SEQ ID NO: 153)(5′-GCGGCCGCCATGGCGTCGAGCGATGAGCG-3′) OO-9R (SEQ ID NO: 154)(5′-GATATCTTACGGGAACGGAGCCAATTTC-3′) For amplification of SEQ ID NO: 73OO-10F (SEQ ID NO: 155) (5′-GCGGCCGCCATGGCGACTCTTAAGGTTTCTG-3′) OO-10R(SEQ ID NO: 156) (5′-AGGCCTTTAAGCATCATCTTCACCGAG-3′) For amplificationof SEQ ID NO: 75 OO-11F (SEQ ID NO: 157)(5′-GCGGCCGCCATGGTGGATCTATTGAACTCG-3′) OO-11R (SEQ ID NO: 158)(5′-AGGCCTTTACAACTCTTGGATATTAAAC-3′) For amplification of SEQ ID NO: 77OO-12F (SEQ ID NO: 159) (5′-GCGGCCGCCATGGCTGGAAAACTCATGCAC-3′) OO-12R(SEQ ID NO: 160) (5′-AGGCCTTTATGGCTCGACAATGATCTTC-3′) For amplificationof SEQ ID NO: 79 pp82F (SEQ ID NO: 49)(5′-ATGGCGCGCCCGACATGAAGCGACGTTGAACG-3′) pp82R (SEQ ID NO: 50)(5′-GCTTAATTAACTTTCCGCAGCCTTCAGGCCGC-3′) For amplification of SEQ ID NO:81 Pk225F (SEQ ID NO: 131) (5′-GGTTAATTAAGGCGCGCCCCCGGAAGCGATGCTGAG-3′)Pk225R (SEQ ID NO: 132) (5′-ATCTCGAGGACGTCCCACAGCCACCGGATTC-3′)

Example 7 Identification of Genes of Interest by Screening ExpressionLibraries with Antibodies

The cDNA clones can be used to produce recombinant protein, for example,in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins arethen normally affinity purified via Ni-NTA affinity chromatography(Qiagen). Recombinant proteins can be used to produce specificantibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni-NTA columnsaturated with the recombinant antigen as described by Gu et al. (1994,BioTechniques 17:257-262). The antibody can then be used to screenexpression cDNA libraries to identify homologous or heterologous genesvia an immunological screening (Sambrook et al., 1989, MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press; orAusubel et al. 1994, “Current Protocols in Molecular Biology”, JohnWiley & Sons).

Example 8 Northern-Hybridization

For RNA hybridization, 20 μg of total RNA or 1 μg of poly-(A)+ RNA wasseparated by gel electrophoresis in 1.25% strength agarose gels usingformaldehyde as described in Amasino (1986, Anal. Biochem. 152:304),transferred by capillary attraction using 10×SSC to positively chargednylon membranes (Hybond N+, Amersham, Braunschweig), immobilized by UVlight, and pre-hybridized for 3 hours at 68° C. using hybridizationbuffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS, 100 μg/ml of herringsperm DNA). The labeling of the DNA probe with the Highprime DNAlabeling kit (Roche, Mannheim, Germany) was carried out during thepre-hybridization using alpha-³²P dCTP (Amersham, Braunschweig,Germany). Hybridization was carried out after addition of the labeledDNA probe in the same buffer at 68° C. overnight. The washing steps werecarried out twice for 15 minutes using 2×SSC and twice for 30 minutesusing 1×SSC, 1% SDS at 68° C. The exposure of the sealed filters wascarried out at −70° C. for a period of 1 day to 14 days.

Example 9 DNA Sequencing and Computational Functional Analysis

The SSH cDNA library as described in Examples 4 and 5 was used for DNAsequencing according to standard methods, in particular by the chaintermination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands).Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (See Sambrook et al. (1989,Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) on a QiageneDNA preparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO: 161 5′-CTAAAGGGAACAAAAGCTG-3′ SEQ IDNO: 162 5′-TGTAAAACGACGGCCAGT-3′ SEQ ID NO: 163

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference see http://pedant.mips.biochem.mpg.de.

The most important algorithms incorporated in EST-MAX are: FASTA: Verysensitive protein sequence database searches with estimates ofstatistical significance (Pearson W.R., 1990, Rapid and sensitivesequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98);BLAST: Very sensitive protein sequence database searches with estimatesof statistical significance (Altschul S. F., Gish W., Miller W., MyersE. W. and Lipman D. J. Basic local alignment search tool. J. Mol. Biol.215:403-410). PREDATOR: High-accuracy secondary structure predictionfrom single and multiple sequences. (Frishman & Argos 1997, 75% accuracyin protein secondary structure prediction. Proteins 27:329-335).CLUSTALW: Multiple sequence alignment (Thompson, J. D., Higgins, D. G.and Gibson, T. J. 1994, CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice, Nucleic AcidsRes. 22:4673-4680). TMAP: Transmembrane region prediction from multiplyaligned sequences (Persson B. & Argos P. 1994, Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments, J. Mol. Biol. 237:182-192). ALOM2:Transmembrane regionprediction from single sequences (Klein P., Kanehisa M., and DeLisi C.1984, Prediction of protein function from sequence properties: Adiscriminant analysis of a database. Biochim. Biophys. Acta 787:221-226.Version 2 by Dr. K. Nakai). PROSEARCH: Detection of PRO SITE proteinsequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M. and Smith J.E. 1992, ProSearch: fast searching of protein sequences with regularexpression patterns related to protein structure and function.Biotechniques 13:919-921). BLIMPS: Similarity searches against adatabase of ungapped blocks (Wallace & Henikoff 1992, PATMAT: Asearching and extraction program for sequence, pattern and block queriesand databases, CABIOS 8:249-254. Written by Bill Alford).

Example 10 Plasmids for Plant Transformation

For plant transformation, various binary vectors such as a pBPS plantbinary vector were used. Construction of the plant binary vectors wasperformed by ligation of the cDNA in sense or antisense orientation intothe vector. In such vectors, a plant promoter was located 5-prime to thecDNA, where it activated transcription of the cDNA; and apolyadenylation sequence was located 3′-prime to the cDNA. Various plantpromoters were used such as a constitutive promoter (Superpromoter), aseed-specific promoter, and a root-specific promoter. Tissue-specificexpression was achieved by using a tissue-specific promoter. Forexample, in some instances, seed-specific expression was achieved bycloning the napin or LeB4 or USP promoter 5-prime to the cDNA. Also, anyother seed specific promoter element can be used, and such promoters arewell known to one of ordinary skill in the art. For constitutiveexpression within the whole plant, in some instances, the Superpromoteror the CaMV 35S promoter was used. The expressed protein also can betargeted to a cellular compartment using a signal peptide, for examplefor plastids, mitochondria, or endoplasmic reticulum (Kermode, 1996,Crit. Rev. Plant Sci. 15:285-423). The signal peptide is cloned 5-primein frame to the cDNA to achieve subcellular localization of the fusionprotein.

The plant binary vectors comprised a selectable marker gene driven underthe control of one of various plant promoters, such as the AtAct2-1promoter and the Nos-promoter; the LMP candidate cDNA under the controlof a root-specific promoter, a seed-specific promoter, a non-tissuespecific promoter, or a constitutive promoter; and a terminator. Partialor full-length LMP cDNA was cloned into the plant binary vector in senseor antisense orientation behind the desired promoter. The recombinantvector containing the gene of interest was transformed into Top10 cells(Invitrogen) using standard conditions. Transformed cells were selectedfor on LB agar containing the selective agent, and cells were grownovernight at 37° C. Plasmid DNA was extracted using the QIAprep SpinMiniprep Kit (Qiagen) following manufacturer's instructions. Analysis ofsubsequent clones and restriction mapping was performed according tostandard molecular biology techniques (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual. 2^(nd) Edition. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y.).

Example 11 Agrobacterium Mediated Plant Transformation

Agrobacterium mediated plant transformation with the LMP nucleic acidsdescribed herein can be performed using standard transformation andregeneration techniques (Gelvin, Stanton B. & Schilperoort R. A, PlantMolecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995in Sect., Ringbuc Zentrale Signatur: BT11-P; Glick, Bernard R. andThompson, John E. Methods in Plant Molecular Biology and Biotechnology,S. 360, CRC Press, Boca Raton 1993). For example, Agrobacterium mediatedtransformation can be performed using the GV3 (pMP90) (Koncz & Schell,1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain.

Arabidopsis thaliana can be grown and transformed according to standardconditions (Bechtold, 1993, Acad. Sci. Paris. 316:1194-1199; Bent etal., 1994, Science 265:1856-1860). Additionally, rapeseed can betransformed with the LMR nucleic acids of the present invention viacotyledon or hypocotyl transformation (Moloney et al., 1989, Plant CellReport 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Useof antibiotics for Agrobacterium and plant selection depends on thebinary vector and the Agrobacterium strain used for transformation.Rapeseed selection is normally performed using kanamycin as selectableplant marker. Additionally, Agrobacterium mediated gene transfer to flaxcan be performed using, for example, a technique described by Mlynarovaet al. (1994, Plant Cell Report 13:282-285).

Transformation of soybean can be performed using for example a techniquedescribed in EP 0424 047, U.S. Pat. No. 5,322,783 (Pioneer Hi-BredInternational) or in EP 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat.No. 5,169,770 (University Toledo). Soybean seeds are surface sterilizedwith 70% ethanol for 4 minutes at room temperature with continuousshaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v)Tween for 20 minutes with continuous shaking. Then the seeds are rinsedfour times with distilled water and placed on moistened sterile filterpaper in a Petri dish at room temperature for 6 to 39 hours. The seedcoats are peeled off, and cotyledons are detached from the embryo axis.The embryo axis is examined to make sure that the meristematic region isnot damaged. The excised embryo axes are collected in a half-opensterile Petri dish and air-dried to a moisture content less than 20%(fresh weight) in a sealed Petri dish until further use.

The method of plant transformation is also applicable to Brassica andother crops. In particular, seeds of canola are surface sterilized with70% ethanol for 4 minutes at room temperature with continuous shaking,followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20minutes, at room temperature with continuous shaking. Then, the seedsare rinsed 4 times with distilled water and placed on moistened sterilefilter paper in a Petri dish at room temperature for 18 hours. The seedcoats are removed and the seeds are air dried overnight in a half-opensterile Petri dish. During this period, the seeds lose approximately 85%of their water content. The seeds are then stored at room temperature ina sealed Petri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LBsolid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin,50 mg/l kanamycin) followed by growth of the single colony in liquid LBmedium to an optical density at 600 nm of 0.8. Then, the bacteriaculture is pelleted at 7000 rpm for 7 minutes at room temperature, andresuspended in MS (Murashige & Skoog, 1962, Physiol. Plant. 15:473-497)medium supplemented with 100 mM acetosyringone. Bacteria cultures areincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately44% moisture content are imbibed for 2 h at room temperature with thepre-induced Agrobacterium suspension culture. (The imbibition of dryembryos with a culture of Agrobacterium is also applicable to maizeembryo axes).

The embryos are removed from the imbibition culture and are transferredto Petri dishes containing solid MS medium supplemented with 2% sucroseand incubated for 2 days, in the dark at room temperature.Alternatively, the embryos are placed on top of moistened (liquid MSmedium) sterile filter paper in a Petri dish and incubated under thesame conditions described above. After this period, the embryos aretransferred to either solid or liquid MS medium supplemented with 500mg/l carbenicillin or 300 mg/l cefotaxime to kill the agrobacteria. Theliquid medium is used to moisten the sterile filter paper. The embryosare incubated during 4 weeks at 25° C., under 440 μmol m⁻²s⁻¹ and 12hours photoperiod. Once the seedlings have produced roots, they aretransferred to sterile metromix soil. The medium of the in vitro plantsis washed off before transferring the plants to soil. The plants arekept under a plastic cover for 1 week to favor the acclimatizationprocess. Then the plants are transferred to a growth room where they areincubated at 25° C., under 440 μmol m⁻²s⁻¹ light intensity and 12 hphotoperiod for about 80 days.

Samples of the primary transgenic plants (T₀) are analyzed by PCR toconfirm the presence of T-DNA. These results are confirmed by Southernhybridization wherein DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labeled probe by PCR as recommended by the manufacturer.

Example 12 In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by incorporationand passage of the plasmid (or other vector) DNA through E. coli orother microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which are impaired in their capabilities to maintain theintegrity of their genetic information. Typical mutator strains havemutations in the genes for the DNA repair system (e.g., mutHLS, mutD,mutT, etc.; for reference, see Rupp W. D. 1996, DNA repair mechanisms,in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)Such strains are well known to those skilled in the art. The use of suchstrains is illustrated, for example, in Greener and Callahan, 1994,Strategies 7:32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 13 Assessment of the mRNA Expression and Activity of aRecombinant Gene Product in the Transformed Organism

The activity of a recombinant gene product in the transformed hostorganism can be measured on the transcriptional level or/and on thetranslational level. A useful method to ascertain the level oftranscription of the gene (an indicator of the amount of mRNA availablefor translation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al. 1988, Current Protocols inMolecular Biology, Wiley: New York), in which a primer designed to bindto the gene of interest is labeled with a detectable tag (usuallyradioactive or chemiluminescent), such that when the total RNA of aculture of the organism is extracted, run on gel, transferred to astable matrix and incubated with this probe, the binding and quantity ofbinding of the probe indicates the presence and also the quantity ofmRNA for this gene. This information at least partially demonstrates thedegree of transcription of the transformed gene. Total cellular RNA canbe prepared from plant cells, tissues or organs by several methods, allwell-known in the art, such as that described in Bornane et al. (1992,Mol. Microbiol. 6:317-326).

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(See, for example, Ausubel et al. 1988, Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or colorimetric label which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

The activity of LMPs that bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such LMP on the expression of othermolecules can be measured using reporter gene assays (such as thatdescribed in Kolmar H. et al., 1995, EMBO J. 14:3895-3904 and referencescited therein). Reporter gene test systems are well known andestablished for applications in both prokaryotic and eukaryotic cells,using enzymes such as beta-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of lipid metabolism membrane-transportproteins can be performed according to techniques such as thosedescribed in Gennis R. B. (1989 Pores, Channels and Transporters, inBiomembranes, Molecular Structure and Function, Springer: Heidelberg,pp. 85-137, 199-234 and 270-322).

Example 14 In Vitro Analysis of the Function of Arabidopsis thaliana andBrassica napus Genes in Transgenic Plants

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M. & Webb, E. C., 1979,Enzymes. Longmans: London; Fersht, 1985, Enzyme Structure and Mechanism.Freeman: New York; Walsh, 1979, Enzymatic Reaction Mechanisms. Freeman:San Francisco; Price, N.C., Stevens, L., 1982, Fundamentals ofEnzymology. Oxford Univ. Press: Oxford; Boyer, P. D., ed. (1983) TheEnzymes, 3rd ed. Academic Press: New York; Bisswanger, H., 1994,Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H. U.,Bergmeyer, J., Grail, M., eds. (1983-1986) Methods of EnzymaticAnalysis, 3rd ed., vol. Verlag Chemie: Weinheim; and Ullmann'sEncyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes. VCH:Weinheim, p. 352-363.

Example 15 Analysis of the Impact of Recombinant LMPs on the Productionof a Desired Seed Storage Compound Fatty Acid Production

The total fatty acid content of Arabidopsis seeds was determined bysaponification of seeds in 0.5 M KOH in methanol at 80° C. for 2 hoursfollowed by LC-MS analysis of the free fatty acids. Total fatty acidcontent of seeds of control and transgenic plants was measured withbulked seeds (usually 5 mg seed weight) of a single plant. Threedifferent types of controls have been used: Col-2 (Columbia-2, theArabidopsis ecotype in which SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:79, or SEQ IDNO:81 has been transformed), Col-0 (Columbia-0, the Arabidopsis ecotypein which SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ: IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, or SEQ ID NO:77 has been transformed), C-24 (an Arabidopsisecotype found to accumulate high amounts of total fatty acids in seeds),and the BPS empty (without an LMP gene of interest) binary vectorconstruct. The controls indicated in the tables below have been grownside by side with the transgenic lines. Differences in the total valuesof the controls are explained either by differences in the growthconditions, which were found to be very sensitive to small variations inthe plant cultivation, or by differences in the standards added toquantify the fatty acid content. Because of the seed bulking, all valuesobtained with T2 seeds, and in part also with T3 seeds, are the resultof a mixture of homozygous (for the gene of interest) and heterozygousevents, implying that these data underestimate the LMP gene effect.

TABLE 5 Determination of the T2 seed total fatty acid content oftransgenic lines of pk123 (containing SEQ ID NO: 1). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.318 ± 0.022 Col-2wild-type control 0.300 ± 0.023 Pk123 transgenic seeds 0.319 ± 0.024Shown are the means (±standard deviation). (Average mean values areshown ± standard deviation, number of individual measurements per plantline: 12-20; Col-2 is the Arabidopsis ecotype the LMP gene has beentransformed in, C-24 is a high-oil Arabidopsis ecotype used as anothercontrol).

TABLE 6 Determination of the T2 seed total fatty acid content oftransgenic lines of pk197 (containing SEQ ID NO: 3). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.371 ± 0.010 Col-2wild-type control 0.353 ± 0.017 Col-2 empty vector control 0.347 ± 0.024Pk197 transgenic seeds 0.366 ± 0.014 Shown are the means (±standarddeviation) of 6 individual plants per line.

TABLE 7 Determination of the T2 seed total fatty acid content oftransgenic lines of pk136 (containing SEQ ID NO: 5). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.351 ± 0.052 Col-2wild-type control 0.344 ± 0.026 Col-2 empty vector control 0.346 ± 0.019Pk136 transgenic seeds 0.374 ± 0.033 Shown are the means (±standarddeviation) of 6 individual plants per line.

TABLE 8 Determination of the T2 seed total fatty acid content oftransgenic lines of pk156 (containing SEQ ID NO: 7). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.400 ± 0.001 Col-2wild-type control 0.369 ± 0.043 Pk156 transgenic seeds 0.389 ± 0.007Shown are the means (±standard deviation) of 6 individual plants perline each.

TABLE 9 Determination of the T2 seed total fatty acid content oftransgenic lines of pk159 (containing SEQ ID NO: 9). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.413 ± 0.019 Col-2wild-type control 0.381 ± 0.019 Pk159 transgenic seeds 0.409 ± 0.008Shown are the means (±standard deviation) of 6 individual plants perline.

TABLE 10 Determination of the T2 seed total fatty acid content oftransgenic lines of pk179 (containing SEQ ID NO: 11). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.400 ± 0.033Col-2 wild-type control 0.339 ± 0.033 Col-2 empty vector control 0.357 ±0.021 Pk179 transgenic seeds 0.384 ± 0.020

TABLE 11 Determination of the T2 seed total fatty acid content oftransgenic lines of pk202 (containing SEQ ID NO: 13). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.413 ± 0.019Col-2 wild-type control 0.381 ± 0.019 Col-2 empty vector control 0.407 ±0.020 Pk202 transgenic seeds 0.426 ± 0.033

TABLE 12 Determination of the T2 seed total fatty acid content oftransgenic lines of pk206 (containing SEQ ID NO: 15). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.422 ± 0.013Col-2 wild-type control 0.354 ± 0.026 Col-2 empty vector control 0.388 ±0.023 Pk206 transgenic seeds 0.414 ± 0.031

TABLE 13 Determination of the T2 seed total fatty acid content oftransgenic lines of pk207 (containing SEQ ID NO: 17). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.371 ± 0.010Col-2 wild-type control 0.353 ± 0.017 Col-2 empty vector control 0.347 ±0.024 Pk207 transgenic seeds 0.370 ± 0.009

TABLE 14 Determination of the T2 seed total fatty acid content oftransgenic lines of pk209 (containing SEQ ID NO: 19). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.400 ± 0.001Col-2 wild-type control 0.369 ± 0.043 Pk209 transgenic seeds 0.397 ±0.007

TABLE 15 Determination of the T2 seed total fatty acid content oftransgenic lines of pk215 (containing SEQ ID NO: 21). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight C-24 wild-type control 0.373 ± 0.045Col-2 wild-type control 0.344 ± 0.026 Col-2 empty vector control 0.346 ±0.019 Pk215 transgenic seeds 0.401 ± 0.014

TABLE 16 Determination of the T3 seed total fatty acid content oftransgenic lines of pk239 (containing SEQ ID NO: 23). Shown are themeans (±standard deviation) of 14-20 individual plants per line.Genotype g total fatty acids/g seed weight C-24 wild-type control 0.334± 0.030 Col-2 empty vector control 0.301 ± 0.027 Pk239-2 transgenicseeds 0.335 ± 0.028 Pk239-9 transgenic seeds 0.335 ± 0.018 Pk239-18transgenic seeds 0.331 ± 0.026 Pk239-20 transgenic seeds 0.343 ± 0.022

TABLE 17 Determination of the T3 seed total fatty acid content oftransgenic lines of pk240 (containing SEQ ID NO: 25). Shown are themeans (±standard deviation) of 10-20 individual plants per line.Genotype g total fatty acids/g seed weight C-24 wild-type control 0.393± 0.037 Col-2 empty vector control 0.342 ± 0.024 Pk240-3 transgenicseeds 0.373 ± 0.033 Pk240-6 transgenic seeds 0.388 ± 0.015 Pk240-10transgenic seeds 0.393 ± 0.025

TABLE 18 Determination of the T2 seed total fatty acid content oftransgenic lines of pk241 (containing SEQ ID NO: 27). Shown are themeans (±standard deviation) of 10 (controls) and 30 (pk241) individualplants per line, respectively. Genotype g total fatty acids/g seedweight Col-2 wild-type control 0.312 ± 0.033 Col-2 empty vector control0.305 ± 0.025 Pk241 transgenic seeds 0.336 ± 0.032

TABLE 19 Determination of the T2 seed total fatty acid content oftransgenic lines of Pk242 (containing SEQ ID NO: 29). Shown are themeans (±standard deviation) of 6 individual plants per line. Genotype gtotal fatty acids/g seed weight Col-2 wild-type control 0.344 ± 0.016Col-2 empty vector control 0.333 ± 0.040 Pk242 transgenic seeds 0.364 ±0.008

TABLE 20 Determination of the T2 seed total fatty acid content oftransgenic lines of Bn011 (containing SEQ ID NO: 31). Shown are themeans (±standard deviation) of 14-20 individual plants per line.Genotype g total fatty acids/g seed weight C-24 wild-type control 0.334± 0.028 Col-2 wild-type control 0.286 ± 0.039 Col-2 empty vector control0.291 ± 0.034 Bn011 transgenic seeds 0.308 ± 0.030

TABLE 21 Determination of the T2 seed total fatty acid content oftransgenic lines of Bn077 (containing SEQ ID NO: 33). Shown are themeans (±standard deviation) of 8-17 individual plants per line. Genotypeg total fatty acids/g seed weight C-24 wild-type control 0.366 ± 0.056Col-2 wild-type control 0.290 ± 0.047 Col-2 empty vector control 0.292 ±0.038 Bn077 transgenic seeds 0.314 ± 0.032

TABLE 22 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb001 (containing SEQ ID NO: 35). Shown are themeans (±standard deviation) of 3 individual control plants and 2individual plants per line. Genotype g total fatty acids/g seed weightCol-0 empty vector control 0.241 ± 0.012 Jb001 transgenic seeds 0.274 ±0.003

TABLE 23 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb002 (containing SEQ ID NO: 37). Shown are themeans (±standard deviation) of 3 individual control plants and 5individual plants per line. Genotype g total fatty acids/g seed weightCol-0 empty vector control 0.191 ± 0.044 Jb002 transgenic seeds 0.273 ±0.020

TABLE 24 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb003 (containing SEQ ID NO: 39). Shown are themeans (±standard deviation) of 3 individual control plants and 2individual plants per line. Genotype g total fatty acids/g seed weightCol-0 empty vector control 0.267 ± 0.011 Jb003 transgenic seeds 0.297 ±0.030

TABLE 25 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb005 (containing SEQ ID NO: 41). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.229 ± 0.021 Jb005transgenic seeds 0.264 ± 0.010 Shown are the means (±standard deviation)of 3 individual control plants and 7 individual plants per line.

TABLE 26 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb007 (containing SEQ ID NO: 43). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.296 ± 0.017 Jb007transgenic seeds 0.320 ± 0.002 Shown are the means (±standard deviation)of 3 individual control plants and 5 individual plants per line.

TABLE 27 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb009 (containing SEQ ID NO: 45). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.227 ± 0.016 Jb009transgenic seeds 0.238 ± 0.004 Shown are the means (±standard deviation)of 3 individual control plants and 3 individual plants per line.

TABLE 28 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb013 (containing SEQ ID NO: 47). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.243 ± 0.011 Jb013transgenic seeds 0.262 ± 0.007 Shown are the means (±standard deviation)of 3 individual control plants and 4 individual plants per line.

TABLE 29 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb017 (containing SEQ ID NO: 51). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.231 ± 0.020 Jb017transgenic seeds 0.269 ± 0.022 Shown are the means (±standard deviation)of 3 individual control plants and 2 individual plants per line.

TABLE 30 Determination of the T2 seed total fatty acid content oftransgenic lines of Jb027 (containing SEQ ID NO: 55). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.235 ± 0.052 Jb027transgenic seeds 0.282 ± 0.014 Shown are the means (±standard deviation)of 3 individual control plants and 2 individual plants per line.

TABLE 31 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-1 (containing SEQ ID NO: 57). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.250 ± 0.009 OO-1transgenic seeds 0.274 ± 0.007 Shown are the means (±standard deviation)of 3 individual control plants and 7 individual plants per line.

TABLE 32 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-4 (containing SEQ ID NO: 63). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.329 ± 0.041 OO-4transgenic seeds 0.380 ± 0.015 Shown are the means (±standard deviation)of 2 individual control plants and 4 individual plants per line.

TABLE 33 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-8 (containing SEQ ID NO: 69). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.379 ± 0.009 OO-8transgenic seeds 0.411 ± 0.008 Shown are the means (±standard deviation)of 4 individual control plants and 2 individual plants per line.

TABLE 34 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-9 (containing SEQ ID NO: 71). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.315 ± 0.020 OO-9transgenic seeds 0.333 ± 0.006 Shown are the means (±standard deviation)of 3 individual control plants and 4 individual plants per line.

TABLE 35 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-11 (containing SEQ ID NO: 75). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.264 ± 0.003 OO-11transgenic seeds 0.278 ± 0.003 Shown are the means (±standard deviation)of 3 individual control plants and 2 individual plants per line.

TABLE 36 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-12 (containing SEQ ID NO: 77). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.290 ± 0.010 OO-12transgenic seeds 0.316 ± 0.008 Shown are the means (±standard deviation)of 3 individual control plants and 9 individual plants per line.

TABLE 37 Determination of the T4 seed total fatty acid content oftransgenic lines of pp82 (containing SEQ ID NO: 79). Genotype g totalfatty acids/g seed weight C-24 wild-type control 0.436 ± 0.050 Col-2wild-type control 0.380 ± 0.020 Col-2 empty vector control 0.378 ± 0.030pp82-15-16 transgenic seeds 0.432 ± 0.040 pp82-15-19 transgenic seeds0.437 ± 0.040 pp82-16-10 transgenic seeds 0.430 ± 0.040 pp82-9-14transgenic seeds 0.449 ± 0.040 Shown are the means (±standard deviation)of 17-20 individual plants per line.

TABLE 38 Determination of the T4 seed total fatty acid content oftransgenic lines of pk225 (containing SEQ ID NO: 81). This particulargene has been down-regulated. Genotype g total fatty acids/g seed weightC-24 wild-type control 0.344 ± 0.048 Col-2 empty vector control 0.327 ±0.031 Pk225-11-19 transgenic seeds 0.350 ± 0.041 Pk225-19-8 transgenicseeds 0.351 ± 0.021 Pk225-7-6 transgenic seeds 0.354 ± 0.037 Pk225-9-10transgenic seeds 0.363 ± 0.042 Shown are the means (±standard deviation)of 17-20 individual plants per line.

TABLE 39 Determination of the T2 seed total fatty acid content oftransgenic lines of OO-3 (containing SEQ ID NO: 61). Genotype g totalfatty acids/g seed weight Col-0 empty vector control 0.365 ± 0.006 OO-3transgenic seeds 0.388 ± 0.006 Shown are the means (±standard deviation)of 4 individual control plants and 6 individual plants per line.

Example 16 Analysis of the Impact of Recombinant Proteins on theProduction of a Desired Seed Storage Compound

The effect of the genetic modification in plants on a desired seedstorage compound (such as a sugar, lipid or fatty acid) can be assessedby growing the modified plant under suitable conditions and analyzingthe seeds or any other plant organ for increased production of thedesired product (i.e., a lipid or a fatty acid). Such analysistechniques are well known to one skilled in the art, and includespectroscopy, thin layer chromatography, staining methods of variouskinds, enzymatic and microbiological methods, and analyticalchromatography such as high performance liquid chromatography (See, forexample, Ullman, 1985, Encyclopedia of Industrial Chemistry, vol. A2,pp. 89-90 and 443-613, VCH: Weinheim; Fallon, A. et al., 1987,Applications of HPLC in Biochemistry in: Laboratory Techniques inBiochemistry and Molecular Biology, vol. 17; Rehm et al., 1993, Productrecovery and purification, Biotechnology, vol. 3, Chapter III, pp.469-714, VCH: Weinheim; Belter, P. A. et al., 1988, Bioseparations:downstream processing for biotechnology, John Wiley & Sons; Kennedy J.F. & Cabral J. M. S., 1992, Recovery processes for biological materials,John Wiley and Sons; Shaeiwitz J. A. & Henry J. D., 1988, Biochemicalseparations in: Ulmann's Encyclopedia of Industrial Chemistry,Separation and purification techniques in biotechnology, vol. B3,Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow F. J. 1989).

Besides the above-mentioned methods, plant lipids are extracted fromplant material as described by Cahoon et al. (1999, Proc. Natl. Acad.Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry442:141-145). Qualitative and quantitative lipid or fatty acid analysisis described in Christie, William W., Advances in Lipid Methodology.Ayr/Scotland: Oily Press.—(Oily Press Lipid Library; Christie, WilliamW., Gas Chromatography and Lipids. A Practical Guide—Ayr, Scotland: OilyPress, 1989 Repr. 1992.—IX, 307 S.—(Oily Press Lipid Library; and“Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)-16 (1977)Progress in the Chemistry of Fats and Other Lipids CODEN.

Unequivocal proof of the presence of fatty acid products can be obtainedby the analysis of transgenic plants following standard analyticalprocedures: GC, GC-MS or TLC as variously described by Christie andreferences therein (1997 in: Advances on Lipid Methodology 4th ed.:Christie, Oily Press, Dundee, pp. 119-169; 1998). Detailed methods aredescribed for leaves by Lemieux et al. (1990, Theor. Appl. Genet.80:234-240) and for seeds by Focks & Benning (1998, Plant Physiol.118:91-101).

Positional analysis of the fatty acid composition at the C-1, C-2 or C-3positions of the glycerol backbone is determined by lipase digestion(See, e.g., Siebertz & Heinz 1977, Z. Naturforsch. 32c:193-205, andChristie, 1987, Lipid Analysis 2^(nd) Edition, Pergamon Press, Exeter,ISBN 0-08-023791-6).

A typical way to gather information regarding the influence of increasedor decreased protein activities on lipid and sugar biosynthetic pathwaysis for example via analyzing the carbon fluxes by labeling studies withleaves or seeds using ¹⁴C-acetate or ¹⁴C-pyruvate (See, e.g. Focks &Benning, 1998, Plant Physiol. 118:91-101; Eccleston & Ohlrogge, 1998,Plant Cell 10:613-621). The distribution of carbon-14 into lipids andaqueous soluble components can be determined by liquid scintillationcounting after the respective separation (for example on TLC plates)including standards like ¹⁴C-sucrose and ¹⁴C-malate (Eccleston &Ohlrogge, 1998, Plant Cell 10:613-621).

Material to be analyzed can be disintegrated via sonification, glassmilling, liquid nitrogen and grinding, or via other applicable methods.The material has to be centrifuged after disintegration. The sediment isresuspended in distilled water, heated for 10 minutes at 100° C., cooledon ice and centrifuged again, followed by extraction in 0.5 M sulfuricacid in methanol containing 2% dimethoxypropane for 1 hour at 90° C.,leading to hydrolyzed oil and lipid compounds resulting intransmethylated lipids. These fatty acid methyl esters are extracted inpetrol ether and finally subjected to GC analysis using a capillarycolumn (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at atemperature gradient between 170° C. and 240° C. for 20 minutes and 5minutes at 240° C. The identity of resulting fatty acid methylesters isdefined by the use of standards available form commercial sources (e.g.,Sigma).

In the case of fatty acids where standards are not available, moleculeidentity is shown via derivatization and subsequent GC-MS analysis. Forexample, the localization of triple bond fatty acids is shown via GC-MSafter derivatization via 4,4-Dimethoxy-oxazolin-Derivaten (Christie,Oily Press, Dundee, 1998).

A common standard method for analyzing sugars, especially starch, ispublished by Stitt M., Lilley R. Mc. C., Gerhardt R. and Heldt M. W.(1989, “Determination of metabolite levels in specific cells andsubcellular compartments of plant leaves,” Methods Enzymol. 74:518-552;for other methods, see also Härtel et al., 1998, Plant Physiol. Biochem.36:407-417 and Focks & Benning, 1998, Plant Physiol. 118:91-101).

For the extraction of soluble sugars and starch, 50 seeds arehomogenized in 500 μl of 80% (v/v) ethanol in a 1.5-ml polypropylenetest tube and incubated at 70° C. for 90 minutes. Followingcentrifugation at 16,000 g for 5 minutes, the supernatant is transferredto a new test tube. The pellet is extracted twice with 500 μl of 80%ethanol. The solvent of the combined supernatants is evaporated at roomtemperature under a vacuum. The residue is dissolved in 50 μl of water,representing the soluble carbohydrate fraction. The pellet left from theethanol extraction, which contains the insoluble carbohydrates includingstarch, is homogenized in 200 μl of 0.2 N KOH, and the suspension isincubated at 95° C. for 1 hour to dissolve the starch. Following theaddition of 35 μl of 1 N acetic acid and centrifugation for 5 minutes at16,000 g, the supernatant is used for starch quantification.

To quantify soluble sugars, 10 μl of the sugar extract is added to 990μl of reaction buffer containing 100 mM imidazole, pH 6.9, 5 mM MgCl₂, 2mM NADP, 1 mM ATP, and 2 units 2 ml⁻¹ of Glucose-6-P-dehydrogenase. Forenzymatic determination of glucose, fructose, and sucrose, 4.5 units ofhexokinase, 1 unit of phosphoglucoisomerase, and 2 μl of a saturatedfructosidase solution are added in succession. The production of NADPHis photometrically monitored at a wavelength of 340 nm. Similarly,starch is assayed in 30 μl of the insoluble carbohydrate fraction with akit from Boehringer Mannheim.

An example for analyzing the protein content in leaves and seeds can befound by Bradford M. M. (1976, “A rapid and sensitive method for thequantification of microgram quantities of protein using the principle ofprotein dye binding,” Anal. Biochem. 72:248-254). For quantification oftotal seed protein, 15-20 seeds are homogenized in 250 μl of acetone ina 1.5-ml polypropylene test tube. Following centrifugation at 16,000 g,the supernatant is discarded and the vacuum-dried pellet is resuspendedin 250 μl of extraction buffer containing 50 mM Tris-HCl, pH 8.0, 250 mMNaCl, 1 mM EDTA, and 1% (w/v) SDS. Following incubation for 2 h at 25°C., the homogenate is centrifuged at 16,000 g for 5 min and 200 ml ofthe supernatant will be used for protein measurements. In the assay,γ-globulin is used for calibration. For protein measurements, Lowry DCprotein assay (Bio-Rad) or Bradford-assay (Bio-Rad) are used.

Enzymatic assays of hexokinase and fructokinase are performedspectrophotometrically according to Renz et al. (1993, Planta190:156-165); enzymatic assays of phosphogluco-isomerase, ATP-dependent6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase,Fructose-1,6-bisphosphate aldolase, triose phosphate isomerase,glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglyceratemutase, enolase and pyruvate kinase are performed according to Burrellet al. (1994, Planta 194:95-101); and enzymatic assays ofUDP-Glucose-pyrophosphorylase according to Zrenner et al. (1995, PlantJ. 7:97-107).

Intermediates of the carbohydrate metabolism, like Glucose-1-phosphate,Glucose-6-phosphate, Fructose-6-phosphate, Phosphoenolpyruvate,Pyruvate, and ATP are measured as described in Härtel et al. (1998,Plant Physiol. Biochem. 36:407-417), and metabolites are measured asdescribed in Jelitto et al. (1992, Planta 188:238-244).

In addition to the measurement of the final seed storage compound (i.e.,lipid, starch or storage protein), it is also possible to analyze othercomponents of the metabolic pathways utilized for the production of adesired seed storage compound, such as intermediates and side-products,to determine the overall efficiency of production of the compound (Fiehnet al., 2000, Nature Biotech. 18:1447-1161).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for alterations in sugar,oil, lipid, or fatty acid contents.

Similarly, plant expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into an appropriate plant cell such as Arabidopsis, soybean,rape, maize, wheat, Medicago truncatula, etc., using standard protocols.The resulting transgenic cells and/or plants derived therefrom can thenbe assayed for alterations in sugar, oil, lipid, or fatty acid contents.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke at al., 1998, Plant J. 15:39-48). The resultant knockoutcells can then be evaluated for their composition and content in seedstorage compounds, and the effect on the phenotype and/or genotype ofthe mutation. For other methods of gene inactivation include U.S. Pat.No. 6,004,804 and Puttaraju et al., 1999, Nature Biotech. 17:246-252).

Example 17 Purification of the Desired Product from TransformedOrganisms

An LMP can be recovered from plant material by various methods wellknown in the art. Organs of plants can be separated mechanically fromother tissue or organs prior to isolation of the seed storage compoundfrom the plant organ. Following homogenization of the tissue, cellulardebris is removed by centrifugation and the supernatant fractioncontaining the soluble proteins is retained for further purification ofthe desired compound. If the product is secreted from cells grown inculture, then the cells are removed from the culture by low-speedcentrifugation, and the supernate fraction is retained for furtherpurification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. Oneskilled in the art would be well-versed in the selection of appropriatechromatography resins and in their most efficacious application for aparticular molecule to be purified. The purified product may beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey J. E. &Ollis D. F., 1986, Biochemical Engineering Fundamentals, McGraw-Hill:New York.

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, analytical chromatography such as high performanceliquid chromatography, NIRS, enzymatic assay, or microbiologically. Suchanalysis methods are reviewed in: Patek et al. (1994, Appl. Environ.Microbiol. 60:133-140), Malakhova et al. (1996, Biotekhnologiya11:27-32), Schmidt et al. (1998, Bioprocess Engineer 19:67-70), Ulmann'sEncyclopedia of Industrial Chemistry (1996, Vol. A27, VCH: Weinheim, p.89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587) andMichal G. (1999, Biochemical Pathways: An Atlas of Biochemistry andMolecular Biology, John Wiley and Sons; Fallon, A. et al. 1987,Applications of HPLC in Biochemistry in: Laboratory Techniques inBiochemistry and Molecular Biology, vol. 17).

Example 18 Screening for Increased Stress Tolerance and Plant Growth

The transgenic plants are screened for their improved stress tolerancedemonstrating that transgene expression confers stress tolerance. Thetransgenic plants are further screened for their growth ratedemonstrating that transgene expression confers increased growth ratesand/or increased seed yield.

Classification of the proteins was done by Blasting against the BLOCKSdatabase (S. Henikoff & J. G. Henikoff, “Protein family classificationbased on searching a database of blocks”, Genomics 19:97-107 (1994)).

Those skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompasses by the claims to the invention disclosedand claimed herein.

APPENDIX A SEQ ID NO: 1, Nucleotide sequence of the open reading frameof Pk123 ATGGCAATCTTCCGAAGTACACTAGTTTTACTGCTGATCCTCTTCTGCCTCACCACTTTTGAGCTTCATGTTCATGCTGCTGAAGATTCACAAGTCGGTGAAGGCGTAGTGAAAATTGATTGCGGTGGGAGATGCAAAGGTAGATGCAGCAAATCGTCGAGGCCAAATCTGTGTTTGAGAGCATGCAACAGCTGTTGTTACCGCTGCAACTGTGTGCCACCAGGCACCGCCGGGAACCACCACCTTTGTCCTTGCTACGCCTCCATTACCACTCGTGGTGGCCGTCTCAAGTGCCCTTAA SEQ ID NO: 2, Deduced amino acid sequence ofthe open reading frame of Pk123MAIFRSTLVLLLILFCLTTFELHVHAAEDSQVGEGVVKIDCGGRCKGRCSKSSRPNLCLRACNSCCYRCNCVPPGTAGNHHLCPCYASITTRGGRLKCP SEQ ID NO: 3, Nucleotidesequence of the open reading frame of Pk197ATGGAGAATGGAGCAACGACGACGAGCACAATTACCATCAAAGGGATTCTGAGTTTGCTAATGGAAAGCATCACAACAGAGGAAGATGAAGGAGGAAAGAGAGTAATATCTCTGGGAATGGGAGACCCAACACTCTACTCGTGTTTTCGTACAACACAAGTCTCTCTTCAAGCTGTTTCTGATTCTCTTCTCTCCAACAAGTTCCATGGTTACTCTCCTACCGTCGGTCTTCCCCAAGCTCGAAGGGCAATAGCAGAGTATCTATCGCGTGATCTTCCATACAAACTTTCACAGGATGATGTGTTTATCACATCGGGTTGCACGCAAGCGATCGATGTAGCATTGTCGATGTTAGCTCGTCCCAGGGCTAATATACTTCTTCCAAGGCCTGGTTTCCCAATCTATGAACTCTGTGCTAAGTTTAGACACCTTGAAGTTCGCTACGTCGATCTTCTTCCGGAAAATGGATGGGAGATCGATCTTGATGCTGTCGAGGCTCTTGCAGACGAAAACACGGTTGCTTTGGTTGTTATAAACCCTGGTAATCCTTGCGGGAATGTCTATAGCTACCAGCATTTGATGAAGATTGCGGAATCGGCGAAAAAACTAGGGTTTCTTGTGATTGCTGATGAGGTTTACGGTCATCTTGCTTTTGGTAGCAAACCGTTTGTGCCAATGGGTGTGTTTGGATCTATTGTTCCTGTGCTTACTCTTGGCTCTTTATCAAAGAGATGGATAGTTCCAGGTTGGCGACTCGGGTGGTTTGTCACCACTGATCCTTCTGGTTCCTTTAAGGACCCTAAGATCATTGAGAGGTTTAAGAAATACTTTGATATTCTTGGTGGACCAGCTACATTTATTCAGGCTGCAGTTCCCACTATTTTGGAACAGACGGATGAGTCTTTCTTCAAGAAAACCTTGAACTCGTTGAAGAACTCTTCGGATATTTGTTGTGACTGGATCAAGGAGATTCCTTGCATTGATTCCTCGCATCGACCAGAAGGATCCATGGCAATGATGGTTAAGCTGAATCTCTCATTACTTGAAGATGTAAGTGACGATATCGACTTCTGTTTCAAGTTAGCTAGGGAAGAATCAGTCATCCTTCTTCCTGGGACCGCGGTGGGGCTGAAGAACTGGCTGAGGATAACGTTTGCAGCAGATGCAACTTCGATTGAAGAAGCTTTTAAAAGGATCAAATGTTTCTATCTTAGACATGCCAAGACTCAATATCCAACCATATAG SEQ ID NO: 4, Deduced aminoacid sequence of the open reading frame of Pk197MENGATTTSTITIKGILSLLMESITTEEDEGGKRVISLGMGDPTLYSCFRTTQVSLQAVSDSLLSNKFHGYSPTVGLPQARRAIAEYLSRDLPYKLSQDDVFITSGCTQAIDVALSMLARPRANILLPRPGFPIYELCAKFRHLEVRYVDLLPENGWEIDLDAVEALADENTVALVVINPGNPCGNVYSYQHLMKIAESAKKLGFLVIADEVYGHLAFGSKPFVPMGVFGSIVPVLTLGSLSKRWIVPGWRLGWFVTTDPSGSFKDPKIIERFKKYFDILGGPATFIQAAVPTILEQTDESFFKKTLNSLKNSSDICCDWIKEIPCIDSSHRPEGSMAMMVKLNLSLLEDVSDDIDFCFKLAREESVILLPGTAVGLKNWLRITFAADATSIEEAFKRIKCFYLRHAK TQYPTI SEQID NO: 5, Nucleotide sequence of the open reading frame of Pk136ATGGCTGAAAAAGTAAAGTCTGGTCAAGTTTTTAACCTATTATGCATATTCTCGATCTTTTTCTTCCTCTTTGTGTTATCAGTGAATGTTTCGGCTGATGTCGATTCTGAGAGAGCGGTGCCATCTGAAGATAAAACGACGACTGTTTGGCTAACTAAAATCAAACGGTCCGGTAAAAATTATTGGGCTAAAGTTAGAGAGACTTTGGATCGTGGACAGTCCCACTTCTTTCCTCCGAACACATATTTTACCGGAAAGAATGATGCGCCGATGGGAGCCGGTGAAAATATGAAAGAGGCGGCGACGAGGAGCTTTGAGCATAGCAAAGCGACGGTGGAGGAAGCTGCTAGATCAGCGGCAGAAGTGGTGAGTGATACGGCGGAAGCTGTGAAAGAAAAGGTGAAGAGGAGCGTTTCCGGTGGAGTGACGCAGCCGTCGGAGGGATCTGAGGAGCTATAA SEQ ID NO: 6, Deduced amino acid sequence of theopen reading frame of Pk136MAEKVKSGQVFNLLCIFSIFFFLFVLSVNVSADVDSERAVPSEDKTTTVWLTKIKRSGKNYWAKVRETLDRGQSHFFPPNTYFTGKNDAPMGAGENMKEAATRSFEHSKATVEEAARSAAEVVSDTAEAVKEKVKRSVSGGVTQPSEGSEEL SEQ ID NO: 7, Nucleotidesequence of the open reading frame of Pk156ATGGCTGGAGAAGAAATAGAGAGGGAGAAGAAATCTGCAGCATCTGCAAGAACTCACACCAGAAACAACACTCAACAAAGTTCTTCTTCTGGTTATCTGAAAACGCTTCTCCTGGTAACGTTCGTCGGAGTTTTAGCATGGGTTTATCAAACAATCCAACCACCACCCGCCAAAATCGTCGGCTCTCCCGGTGGACCCACCGTGACATCACCGAGGATCAAACTGAGAGACGGAAGACATCTGGCTTACACAGAATTCGGAATCCCTAGAGACGAAGCCAAGTTCAAGATCATAAACATCCACGGCTTCGATTCTTGTATGCGAGACTCGCATTTCGCCAATTTCTTATCGCCGGCTCTTGTGGAGGAATTGAGGATATACATTGTGTCTTTTGATCGTCCTGGTTATGGAGAGAGTGATCCTAACCTGAATGGGTCACCAAGAAGCATAGCATTGGATATAGAAGAGCTTGCTGATGGGTTAGGACTAGGACCTCAGTTCTATCTCTTTGGTTACTCCATGGGTGGTGAAATTACATGGGCATGCCTTAACTACATTCCTCACAGGTTAGCAGGAGCTGCCCTTGTAGCTCCAGCGATTAACTATTGGTGGAGAAACTTACCGGGAGATTTAACAAGAGAAGCTTTCTCTCTTATGCATCCTGCAGATCAATGGTCACTTCGAGTAGCTCATTATGCTCCTTGGCTTACATATTGGTGGAACACTCAGAAATGGTTCCCAATCTCCAATGTGATTGCCGGTAATCCCATTATTTTCTCACGTCAGGACATGGAGATCTTGTCGAAGCTCGGATTCGTCAATCCAAATCGGGCATACATAAGACAACAAGGTGAATATGTAAGCTTACACCGAGATTTGAATGTCGCATTTTCAAGCTGGGAGTTTGATCCGTTAGACCTTCAAGATCCGTTCCCGAACAACAATGGCTCAGTTCACGTATGGAATGGCGATGAGGATAAGTTTGTGCCAGTAAAGCTTCAACGGTATGTCGCGTCAAAGCTGCCATGGATTCGTTACCATGAAATATCTGGATCAGGACATTTTGTACCATTTGTGGAAGGTATGACTGATAAGATCATCAAGTCACTTTTGGTTGGGGAAGAAGATGTAAGTGAGAGTAGAGAAGCCT CTGTTTAA SEQ IDNO: 8, Deduced amino acid sequence of the open reading frame of Pk156MAGEEIEREKKSAASARTHTRNNTQQSSSSGYLKTLLLVTFVGVLAWVYQTIQPPPAKIVGSPGGPTVTSPRIKLRDGRHLAYTEFGIPRDEAKFKIINIHGFDSCMRDSHFANFLSPALVEELRIYIVSFDRPGYGESDPNLNGSPRSIALDIEELADGLGLGPQFYLFGYSMGGEITWACLNYIPHRLAGAALVAPAINYWWRNLPGDLTREAFSLMHPADQWSLRVAHYAPWLTYWWNTQKWFPISNVIAGNPIIFSRQDMEILSKLGFVNPNRAYIRQQGEYVSLHRDLNVAFSSWEFDPLDLQDPFPNNNGSVHVWNGDEDKFVPVKLQRYVASKLPWIRYHEISGSGHFVPFVEGMTDKIIKSLLVGEEDVSESREASV SEQ ID NO: 9, Nucleotidesequence of the open reading frame of Pk159ATGGCTGGAGTGATGAAGTTGGCATGCATGGTCTTGGCTTGCATGATTGTGGCCGGTCCAATCACAGCGAACGCGCTTATGAGTTGTGGCACCGTCAACGGCAACCTGGCAGGGTGCATTGCCTACTTGACCCGAGGTGCTCCACTTACCCAAGGGTGCTGCAACGGCGTTACTAACCTTAAAAACATGGCCAGTACAACCCCAGACCGTCAGCAAGCTTGCCGTTGCCTTCAATCTGCCGCTAAAGCCGTTGGTCCCGGTCTCAACACTGCCCGTGCAGCTGGACTTCCTAGCGCATGCAAAGTCAATATTCCTTACAAAATCAGCGCCAGCACCAACTGCAACACCGTGAGGTGA SEQ ID NO: 10, Deduced amino acid sequenceof the open reading frame of Pk159MAGVMKLACMVLACMIVAGPITANALMSCGTVNGNLAGCIAYLTRGAPLTQGCCNGVTNLKNMASTTPDRQQACRCLQSAAKAVGPGLNTARAAGLPSACKVNIPYKISAS TNCNTVR SEQ IDNO: 11, Nucleotide sequence of the open reading frame of Pk179ATGGGGCTTGCTGTGGTGGACAAAAACACAGTTGCGATTTCTGCATCTGATGTTATGTTGTCCTTTGCTGCTTTTCCAGTCGAGATTCCTGGAGAGGTAGTATTTCTTCATCCCGTTCACAACTATGCTCTGATTGCGTATAATCCATCAGCAATGGATCCTGCCAGTGCTTCAGTCATTCGTGCAGCTGAGCTACTACCTGAACCTGCACTCCAACGTGGAGATTCAGTCTATCTTGTCGGATTGAGTAGGAACCTTCAAGCTACATCAAGAAAATCTATTGTAACCAATCCATGTGCAGCGTTAAACATTGGTTCTGCTGATTCTCCCCGTTACAGAGCTACTAATATGGAAGTAATTGAGCTTGATACAGATTTTGGTAGCTCATTTTCAGGGGCGCTGACTGATGAGCAGGGAAGAATTCGGGCTATTTGGGGAAGTTTTTCGACTCAGGTTAAATATAGTTCCACTTCTTCAGAAGACCACCAGTTTGTCAGAGGTATCCCAGTATATGCAATCAGCCAAGTCCTTGAAAAAATCATAACCGGTGGAAATGGACCAGCTCTTCTCATAAATGGTGTCAAAAGGCCAATGCCACTTGTTCGGATTTTGGAAGTTGAATTGTATCCTACTTTGCTTTCAAAAGCCCGGAGTTTTGGTCTGAGTGATGAATGGATCCAAGTCCTAGTCAAGAAGGATCCTGTTAGACGTCAAGTTCTGCGTGTTAAAGGTTGCCTGGCAGGATCAAAAGCTGAAAACCTTCTTGAACAAGGCGATATGGTTCTGGCAGTCAATAAGATGCCAGTTACATGCTTCAATGACATAGAAGCTGCTTGCCAAACATTGGATAAGGGTAGTTACAGCGATGAAAATCTCAATCTAACAATCCTTAGACAGGGCCAAGAACTGGAGCTCGTAGTTGGAACTGATAAGAGAGATGGGAATGGAACGACAAGAGTGATAAATTGGTGCGGATGCGTTGTTCAGGATCCTCATCCTGCGGTTCGTGCTCTTGGATTTCTTCCTGAGGAAGGTCATGGTGTCTATGTCACAAGATGGTGTCACGGGAGTCCCGCTCACCGATATGGCCTCTACGCGCTTCAATGGATCGTGGAAGTTAATGGGAAGAAGACTCCTGACCTAAACGCATTCGCAGATGCTACCAAGGAGCTAGAACACGGGCAGTTTGTGCGTATTAGGACTGTTCATCTAAACGGCAAGCCACGAGTATTGACCCTGAAACAAGATCTCCATTACTGGCCGACTTGGGAATTGAGGTTCGACCCAGAGACTGCTCTTTGGCGGAGAAATATATTGA AAGCCTTGCAGTAASEQ ID NO: 12, Deduced amino acid sequence of the open reading frame ofPk179 MGLAVVDKNTVAISASDVMLSFAAFPVEIPGEVVFLHPVHNYALIAYNPSAMDPASASVIRAAELLPEPALQRGDSVYLVGLSRNLQATSRKSIVTNPCAALNIGSADSPRYRATNMEVIELDTDFGSSFSGALTDEQGRIRAIWGSFSTQVKYSSTSSEDHQFVRGIPVYAISQVLEKIITGGNGPALLINGVKRPMPLVRILEVELYPTLLSKARSFGLSDEWIQVLVKKDPVRRQVLRVKGCLAGSKAENLLEQGDMVLAVNKMPVTCFNDIEAACQTLDKGSYSDENLNLTILRQGQELELVVGTDKRDGNGTTRVINWCGCVVQDPHPAVRALGFLPEEGHGVYVTRWCHGSPAHRYGLYALQWIVEVNGKKTPDLNAFADATKELEHGQFVRIRTVHLNGKPRVLTLKQDLHYWPTWELRFDPETALWRRNILKALQ SEQ ID NO: 13, Nucleotidesequence of the open reading frame of Pk202ATGGCGTTCACGGCGCTTGTGTTCATTGTGTTCGTGGTGGGTGTCATGGTTTCTCCAGTTTCAATCAGAGCAACTGAGGTCAAACTTTCTGGAGGAGAAGCTGATGTAACGTGTGATGCAGTACAGCTTAGTTCATGCGCAACACCAATGCTCACAGGAGTACCACCGTCTACAGAGTGTTGCGGGAAACTGAAGGAGCAACAGCCGTGTTTTTGTACATATATTAAAGATCCAAGATATAGTCAATATGTTGGTTCTGCAAATGCTAAGAAAACGTTAGCAACTTGTGGTGTTCCTTATCCTACTTGTTGA SEQ ID NO: 14, Deduced amino acidsequence of the open reading frame of Pk202MAFTALVFIVFVVGVMVSPVSIRATEVKLSGGEADVTCDAVQLSSCATPMLTGVPPSTECCGKLKEQQPCFCTYIKDPRYSQYVGSANAKKTLATCGVPYPTC SEQ ID NO: 15, Nucleotidesequence of the open reading frame of Pk206ATGGCCCTTGATGAGCTTCTCAAGACTGTCTTGCCACCAGCTGAGGAAGGGCTTGTTCGTCAGGGAAGCTTGACGTTACCTCGAGATCTCAGTAAAAAGACAGTTGATGAGGTCTGGAGAGATATCCAACAGGACAAGAATGGAAACGGTACTAGTACTACTACTACTCATAAGCAGCCTACACTCGGTGAAATAACACTTGAGGATTTGTTGTTGAGAGCTGGTGTAGTGACTGAGACAGTAGTCCCTCAAGAAAATGTTGTTAACATAGCTTCAAATGGGCAATGGGTTGAGTATCATCATCAGCCTCAACAACAACAAGGGTTTATGACATATCCGGTTTGCGAGATGCAAGATATGGTGATGATGGGTGGATTATCGGATACACCACAAGCGCCTGGGAGGAAAAGAGTAGCTGGAGAGATTGTGGAGAAGACTGTTGAGAGGAGACAGAAGAGGATGATCAAGAACAGAGAATCTGCAGCACGTTCACGAGCTAGGAAACAGGCTTATACACATGAATTAGAGATCAAGGTTTCAAGGTTAGAAGAAGAAAACGAAAAACTTCGGAGGCTAAAGGAGGTGGAGAAGATCCTACCAAGTGAACCACCACCAGATCCTAAGTGGAAGCTCCGGCGAACAAACTCTGCT TCTCTCTGA SEQ IDNO: 16, Deduced amino acid sequence of the open reading frame of Pk206MALDELLKTVLPPAEEGLVRQGSLTLPRDLSKKTVDEVWRDIQQDKNGNGTSTTTTHKQPTLGEITLEDLLLRAGVVTETVVPQENVVNIASNGQWVEYHHQPQQQQGFMTYPVCEMQDMVMMGGLSDTPQAPGRKRVAGEIVEKTVERRQKRMIKNRESAARSRARKQAYTHELEIKVSRLEEENEKLRRLKEVEKILPSEPPPDPKWKLRRTNSASL SEQ ID NO: 17,Nucleotide sequence of the open reading frame of Pk207ATGGCGCAATCCCGATTATTAGCGTTTGCTTCAGCGGCGCGTTCACGTGTTCGACCAATCGCTCAAAGGCGTTTAGCGTTTGGATCATCCACGTCTGGTCGCACAGCTGATCCAGAGATCCATGCCGGTAACGATGGAGCCGATCCAGCTATCTATCCGAGAGACCCTGAAGGTATGGATGATGTTGCAAACCCTAAAACGGCGGCGGAAGAAATCGTAGACGATACTCCCCGACCGAGTTTAGAAGAGCAACCGCTTGTACCGCCGAAATCTCCACGCGCCACTGCGCACAAGCTAGAGAGTACTCCCGTTGGTCACCCGTCAGAACCTCATTTCCAACAGAAACGAAAAAACTCCACCGCTTCTCCGCCGTCGCTTGATTCCGTGAGCTGTGCTGGTTTAGACGGTTCACCATGGCCGAGAGACGAAGGAGAAGTGGAAGAGCAAAGGCGAAGAGAAGATGAAACAGAGAGTGACCAAGAGTTTTACAAACACCACAAAGCTTCTCCGTTATCGGAGATTGAATTCGCCGATACTCGGAAACCTATTACGCAAGCTACCGATGGAACTGCCTACCCAGCCGGGAAAGATGTGATCGGATGGTTACCGGAGCAGCTAGACACGGCGGAAGAATCTTTGATGAAAGCAACAATGATATTCAAACGCAACGCAGAACGTGGCGATCCTGAAACGTTTCCTCATTCTAGAATCTTAAGAGAAATGAGAGGCGAGTGGTTTTAA SEQ ID NO: 18, Deduced amino acidsequence of the open reading frame of Pk207MAQSRLLAFASAARSRVRPIAQRRLAFGSSTSGRTADPEIHAGNDGADPAIYPRDPEGMDDVANPKTAAEEIVDDTPRPSLEEQPLVPPKSPRATAHKLESTPVGHPSEPHFQQKRKNSTASPPSLDSVSCAGLDGSPWPRDEGEVEEQRRREDETESDQEFYKHHKASPLSEIEFADTRKPITQATDGTAYPAGKDVIGWLPEQLDTAEESLMKATMIFKRNAERGDPETFPHSRILREMRGEWF SEQ ID NO: 19, Nucleotide sequence of the open readingframe of Pk209 ATGTCCGTGGCTCGATTCGATTTCTCTTGGTGCGATGCTGATTATCACCAGGAGACGCTGGAGAATCTGAAGATAGCTGTGAAGAGCACTAAGAAGCTTTGTGCTGTTATGCTAGACACTGTAGGACCTGAGTTGCAAGTTATTAACAAGACTGAGAAAGCTATTTCTCTTAAAGCTGATGGCCTTGTAACTTTGACTCCGAGTCAAGATCAAGAAGCCTCCTCTGAAGTCCTTCCCATTAATTTTGATGGGTTAGCGAAGGCGGTTAAGAAAGGAGACACTATCTTTGTTGGACAATACCTCTTCACTGGTAGTGAAACAACTTCAGTTTGGCTTGAGGTTGAAGAAGTTAAAGGAGATGATGTCATTTGTATTTCAAGGAATGCTGCTACTCTGGGTGGTCCGTTATTCACATTGCACGTCTCTCAAGTTCACATTGATATGCCAACCCTAACTGAGAAGGATAAGGAGGTTATAAGTACATGGGGAGTTCAGAATAAGATCGACTTTCTCTCATTATCTTATTGTCGACATGCAGAAGATGTTCGCCAGGCCCGTGAGTTGCTTAACAGTTGTGGTGACCTCTCTCAAACACAAATATTTGCGAAGATTGAGAATGAAGAGGGACTAACCCACTTTGACGAAATTCTACAAGAAGCAGATGGCATTATTCTTTCTCGTGGGAATTTGGGTATCGATCTACCTCCGGAAAAGGTGTTTTTGTTCCAAAAGGCTGCTCTTTACAAGTGTAACATGGCTGGAAAGCCTGCCGTTCTTACTCGTGTTGTAGACAGTATGACAGACAATCTGCGGCCAACTCGTGCAGAGGCAACTGATGTTGCTAATGCTGTTTTAGATGGAAGTGATGCAATTCTTCTTGGTGCTGAGACTCTTCGTGGATTGTACCCTGTTGAAACCATATCAACTGTTGGTAGAATCTGTTGTGAGGCAGAGAAAGTTTTCAACCAAGATTTGTTCTTTAAGAAGACTGTCAAGTATGTTGGAGAACCAATGACTCACTTGGAATCTATTGCTTCTTCTGCTGTACGGGCAGCAATCAAGGTTAAGGCATCCGTAATTATATGCTTCACCTCGTCTGGCAGAGCAGCAAGGTTGATTGCCAAATACCGTCCAACTATGCCCGTTCTCTCTGTTGTCATTCCCCGACTTACGACAAATCAGCTGAAGTGGAGCTTTAGCGGAGCCTTTGAGGCAAGGCAGTCACTTATTGTCAGAGGTCTTTTCCCCATGCTTGCTGATCCTCGTCACCCTGCGGAATCAACAAGTGCAACAAATGAGTCGGTTCTTAAAGTGGCTCTAGACCATGGGAAGCAAGCCGGAGTGATCAAGTCACATGACAGAGTTGTGGTCTGTCAGAAAGTGGGAGATGCGTCCGTGGTCAAAATCATCGAGCTAGA GGATTAG SEQ IDNO: 20, Deduced amino acid sequence of the open reading frame of Pk209MSVARFDFSWCDADYHQETLENLKIAVKSTKKLCAVMLDTVGPELQVINKTEKAISLKADGLVTLTPSQDQEASSEVLPINFDGLAKAVKKGDTIFVGQYLFTGSETTSVWLEVEEVKGDDVICISRNAATLGGPLFTLHVSQVHIDMPTLTEKDKEVISTWGVQNKIDFLSLSYCRHAEDVRQARELLNSCGDLSQTQIFAKIENEEGLTHFDEILQEADGIILSRGNLGIDLPPEKVFLFQKAALYKCNMAGKPAVLTRVVDSMTDNLRPTRAEATDVANAVLDGSDAILLGAETLRGLYPVETISTVGRICCEAEKVFNQDLFFKKTVKYVGEPMTHLESIASSAVRAAIKVKASVIICFTSSGRAARLIAKYRPTMPVLSVVIPRLTTNQLKWSFSGAFEARQSLIVRGLFPMLADPRHPAESTSATNESVLKVALDHGKQAGVIKSHDRVVVCQKVGDASVVKIIELED SEQ ID NO: 21, Nucleotide sequence of the open readingframe of Pk215 ATGGCGATTTACAGATCTCTAAGAAAGCTAGTTGAAATCAATCACCGGAAAACAAGACCATTCCTCACCGCCGCTACAGCTTCCGGCGGAACCGTTTCTCTGACTCCACCGCAGTTTTCGCCGTTGTTCCCACATTTCTCACACCGTTTATCTCCGCTTTCGAAATGGTTCGTTCCTCTTAATGGACCTCTCTTCTTATCTTCTCCTCCTTGGAAACTTCTCCAGTCTGCGACACCTTTGCACTGGCGCGGAAACGGCTCTGTTTTGAAAAAAGTCGAAGCTCTGAATCTTAGATTGGATCGAATTAGAAGCAGAACTAGGTTTCCGAGACAGTTAGGGTTACAGTCTGTGGTACCAAACATATTGACGGTGGATCGCAACGATTCCAAGGAAGAAGATGGTGGAAAATTAGTCAAGAGTTTTGTTAATGTGCCGAATATGATATCAATGGCGAGATTAGTATCTGGTCCTGTGCTTTGGTGGATGATCTCGAATGAGATGTATTCTTCTGCTTTCTTAGGGTTGGCTGTTTCTGGAGCTAGTGATTGGTTAGATGGTTACGTGGCTCGGAGGATGAAGATTAACTCTGTGGTTGGCTCGTACCTTGATCCTCTTGCAGACAAGGTTCTTATCGGGTGTGTAGCAGTAGCAATGGTGCAGAAGGATCTCTTACATCCTGGACTGGTTGGAATTGTGTTGTTACGGGATGTTGCACTCGTTGGTGGTGCAGTTTACCTAAGGGCACTAAACTTGGACTGGAGGTGGAAAACTTGGAGTGACTTCTTCAATCTAGATGGTTCAAGTCCTCAGAAAGTAGAACCATTGTTTATAAGCAAGGTGAATACAGTTTTCCAGTTGACTCTAGTCGCTGGTGCAATACTTCAACCAGAGTTTGGGAATCCAGACACCCAGACATGGATCACTTATCTAAGGTAA SEQ ID NO: 22,Deduced amino acid sequence of the open reading frame of Pk215MAIYRSLRKLVEINHRKTRPFLTAATASGGTVSLTPPQFSPLFPHFSHRLSPLSKWFVPLNGPLFLSSPPWKLLQSATPLHWRGNGSVLKKVEALNLRLDRIRSRTRFPRQLGLQSVVPNILTVDRNDSKEEDGGKLVKSFVNVPNMISMARLVSGPVLWWMISNEMYSSAFLGLAVSGASDWLDGYVARRMKINSVVGSYLDPLADKVLIGCVAVAMVQKDLLHPGLVGIVLLRDVALVGGAVYLRALNLDWRWKTWSDFFNLDGSSPQKVEPLFISKVNTVFQLTLVAGAILQPEFGNPDTQTWITYLR SEQ ID NO: 23, Nucleotide sequence of theopen reading frame of Pk239ATGGTAAAGGAAACTCTAATTCCTCCGTCATCTACGTCAATGACGACCGGAACATCTTCTTCTTCGTCTCTTTCAATGACGTTATCCTCAACAAACGCGTTATCGTTTTTGTCGAAAGGATGGAGAGAGGTATGGGATTCAGCAGATGCGGATTTGCAGCTGATGCGAGACAGAGCTAACTCTGTTAAGAATCTAGCATCAACGTTCGATAGAGAGATCGAGAATTTCCTCAATAACTCGGCGAGGTCTGCGTTTCCCGTTGGTTCACCATCGGCGTCGTCTTTCTCAAATGAAATTGGTATCATGAAGAAGCTTCAGCCGAAGATTTCGGAGTTTCGTAGGGTTTATTCGGCGCCGGAGATTAGTCGCAAGGTTATGGAGAGATGGGGACCTGCGAGAGCGAAGCTTGGAATGGATCTATCGGCGATTAAGAAGGCGATTGTGTCTGAGATGGAATTGGATGAGCGTCAGGGAGTTTTGGAGATGAGTAGATTGAGGAGACGGCGTAATAGTGATAGGGTTAGGTTTACGGAGTTTTTCGCGGAGGCTGAGAGAGATGGAGAAGCTTATTTCGGTGATTGGGAACCGATTAGGTCTTTGAAGAGTAGATTTAAAGAGTTTGAGAAACGAAGCTCGTTAGAAATATTGAGTGGATTCAAGAACAGTGAATTTGTTGAGAAGCTCAAAACCAGCTTTAAATCAATTTACAAAGAAACTGATGAGGCTAAGGATGTCCCTCCGTTGGATGTACCTGAACTGTTGGCATGTTTGGTTAGACAATCTGAACCTTTTCTTGATCAGATTGGTGTTAGAAAGGATACATGTGACCGAATAGTAGAAAGCCTTTGCAAATGCAAGAGCCAACAACTTTGGCGTCTGCCATCTGCACAAGCATCCGATTTAATTGAAAATGATAACCATGGAGTTGATTTGGATATGAGGATAGCCAGTGTTCTTCAAAGCACAGGACACCATTATGATGGTGGGTTTTGGACTGATTTTGTGAAGCCTGAGACACCGGAAAACAAAAGGCATGTGGCAATTGTTACAACAGCTAGTCTTCCTTGGATGACCGGAACAGCTGTAAATCCGCTATTCAGAGCGGCGTATTTGGCAAAAGCTGCAAAACAGAGTGTTACTCTCGTGGTTCCTTGGCTCTGCGAATCTGATCAAGAACTAGTGTATCCAAACAATCTCACCTTCAGCTCACCTGAAGAACAAGAGAGTTATATACGTAAATGGTTGGAGGAAAGGATTGGTTTCAAGGCTGATTTTAAAATCTCCTTTTACCCAGGAAAGTTTTCAAAAGAAAGGCGCAGCATATTTCCTGCTGGTGACACTTCTCAATTTATATCGTCAAAAGATGCTGACATTGCTATACTTGAAGAACCTGAACATCTCAACTGGTATTATCACGGCAAGCGTTGGACTGATAAATTCAACCATGTTGTTGGAATTGTCCACACAAACTACTTAGAGTACATCAAGAGGGAGAAGAATGGAGCTCTTCAAGCATTTTTTGTGAACCATGTAAACAATTGGGTCACACGAGCGTATTGTGACAAGGTTCTTCGCCTCTCTGCGGCAACACAAGATTTACCAAAGTCTGTTGTATGCAATGTCCATGGTGTCAATCCCAAGTTCCTTATGATTGGGGAGAAAATTGCTGAAGAGAGATCCCGTGGTGAACAAGCTTTCTCAAAAGGTGCATACTTCTTAGGAAAAATGGTGTGGGCTAAAGGATACAGAGAACTAATAGATCTGATGGCTAAACACAAAAGCGAACTTGGGAGCTTCAATCTAGATGTATATGGGAACGGTGAAGATGCAGTCGAGGTCCAACGTGCAGCAAAGAAACATGACTTGAATCTCAATTTCCTCAAAGGAAGGGACCACGCTGACGATGCTCTTCACAAGTACAAAGTGTTCATAAACCCCAGCATCAGCGATGTTCTATGCACAGCAACCGCAGAAGCACTAGCCATGGGGAAGTTTGTGGTGTGTGCAGATCACCCTTCAAACGAATTCTTTAGATCATTCCCGAACTGCTTAACTTACAAAACATCCGAAGACTTTGTGTCCAAAGTGCAAGAAGCAATGACGAAAGAGCCACTACCTCTCACTCCTGAACAAATGTACAATCTCTCTTGGGAAGCAGCAACACAGAGGTTCATGGAGTATTCAGATCTCGATAAGATCTTAAACAATGGAGAGGGAGGAAGGAAGATGCGAAAATCAAGATCGGTTCCGAGCTTTAACGAGGTGGTCGATGGAGGATTGGCATTCTCACACTATGTTCTAACAGGGAACGATTTCTTGAGACTATGCACTGGAGCAACACCAAGAACAAAAGACTATGATAATCAACATTGCAAGGATCTGAATCTCGTACCACCTCACGTTCACAAGCCAATCTTCGGCTGGTAG SEQ ID NO: 24, Deduced amino acid sequence ofthe open reading frame of Pk239MVKETLIPPSSTSMTTGTSSSSSLSMTLSSTNALSFLSKGWREVWDSADADLQLMRDRANSVKNLASTFDREIENFLNNSARSAFPVGSPSASSFSNEIGIMKKLQPKISEFRRVYSAPEISRKVMERWGPARAKLGMDLSAIKKAIVSEMELDERQGVLEMSRLRRRRNSDRVRFTEFFAEAERDGEAYFGDWEPIRSLKSRFKEFEKRSSLEILSGFKNSEFVEKLKTSFKSIYKETDEAKDVPPLDVPELLACLVRQSEPFLDQIGVRKDTCDRIVESLCKCKSQQLWRLPSAQASDLIENDNHGVDLDMRIASVLQSTGHHYDGGFWTDFVKPETPENKRHVAIVTTASLPWMTGTAVNPLFRAAYLAKAAKQSVTLVVPWLCESDQELVYPNNLTFSSPEEQESYIRKWLEERIGFKADFKISFYPGKFSKERRSIFPAGDTSQFISSKDADIAILEEPEHLNWYYHGKRWTDKFNHVVGIVHTNYLEYIKREKNGALQAFFVNHVNNWVTRAYCDKVLRLSAATQDLPKSVVCNVHGVNPKFLMIGEKIAEERSRGEQAFSKGAYFLGKMVWAKGYRELIDLMAKHKSELGSFNLDVYGNGEDAVEVQRAAKKHDLNLNFLKGRDHADDALHKYKVFINPSISDVLCTATAEALAMGKFVVCADHPSNEFFRSFPNCLTYKTSEDFVSKVQEAMTKEPLPLTPEQMYNLSWEAATQRFMEYSDLDKILNNGEGGRKMRKSRSVPSFNEVVDGGLAFSHYVLTGNDFLRLCTGATPRTKDYDNQHCKDLNL VPPHVHKPIFGWSEQ ID NO: 25, Nucleotide sequence of the open reading frame of Pk240ATGGCGACTTTTGCTGAACTTGTTTTATCGACTTCTCGCTGTACATGCCCTTGCCGTTCATTCACTAGAAAACCCCTAATTCGTCCCCCTTTATCTGGTCTGCGTCTCCCCGGTGATACCAAACCATTGTTTCGTTCCGGACTTGGTCGGATTTCTGTTAGCCGGCGTTTCCTCACGGCCGTTGCTCGAGCTGAATCAGACCAGCTTGGTGATGATGACCACTCAAAGGGAATTGATAGAATCCATAACTTGCAGAATGTGGAAGATAAGCAGAAGAAAGCAAGCCAGCTTAAGAAAAGAGTGATCTTTGGTATTGGCATTGGTTTACCTGTTGGATGTGTTGTGTTAGCTGGAGGATGGGTTTTCACTGTAGCTTTAGCATCTTCTGTTTTTATCGGTTCCCGCGAATATTTCGAGCTTGTTAGAAGTAGAGGCATAGCTAAAGGAATGACTCCTCCTCCACGATATGTATCTCGAGTTTGCTCGGTTATATGTGCCCTTATGCCCATACTTACACTGTACTTTGGTAACATTGATATATTGGTGACATCTGCAGCATTTGTTGTTGCAATAGCATTGTTAGTACAAAGAGGATCCCCACGTTTTGCTCAGCTGAGTAGTACAATGTTTGGTCTGTTTTACTGTGGTTATCTCCCTTCTTTCTGGGTTAAGCTTCGCTGTGGTTTAGCTGCTCCTGCGCTTAACACTGGTATCGGAAGGACATGGCCAATTCTTCTTGGTGGTCAAGCTCATTGGACAGTTGGACTTGTGGCAACATTGATTTCTTTCAGCGGTGTAATTGCGACAGACACATTTGCTTTTCTCGGTGGAAAGACTTTTGGTAGGACACCTCTTACTAGTATTAGTCCCAAGAAGACATGGGAAGGAACTATTGTAGGACTTGTTGGTTGTATAGCCATTACCATATTACTCTCTAAATATCTCAGTTGGCCACAATCTCTGTTCAGCTCAGTAGCTTTTGGGTTTCTTAACTTCTTTGGGTCAGTCTTTGGTGATCTTACTGAATCAATGATCAAGCGTGATGCTGGCGTCAAAGACTCTGGTTCACTTATCCCAGGACACGGTGGAATATTAGATAGAGTTGATAGTTACATTTTCACCGGCGCATTAGCTTATTCATTCATCAAAACATCCCTAAAACTTT ACGGAGTTTGA SEQID NO: 26, Deduced amino acid sequence of the open reading frame ofPk240 MATFAELVLSTSRCTCPCRSFTRKPLIRPPLSGLRLPGDTKPLFRSGLGRISVSRRFLTAVARAESDQLGDDDHSKGIDRIHNLQNVEDKQKKASQLKKRVIFGIGIGLPVGCVVLAGGWVFTVALASSVFIGSREYFELVRSRGIAKGMTPPPRYVSRVCSVICALMPILTLYFGNIDILVTSAAFVVAIALLVQRGSPRFAQLSSTMFGLFYCGYLPSFWVKLRCGLAAPALNTGIGRTWPILLGGQAHWTVGLVATLISFSGVIATDTFAFLGGKTFGRTPLTSISPKKTWEGTIVGLVGCIAITILLSKYLSWPQSLFSSVAFGFLNFFGSVFGDLTESMIKRDAGVKDSGSLIPGHGGILDRVDSYIFTGALAYSFIKTSLKLYGV SEQ ID NO: 27, Nucleotidesequence of the open reading frame of Pk241ATGGCTCAAACCATGCTGCTTACTTCAGGCGTCACCGCCGGCCATTTTTTGAGGAACAAGAGCCCTTTGGCTCAGCCCAAAGTTCACCATCTCTTCCTCTCTGGAAACTCTCCGGTTGCACTACCATCTAGGAGACAATCATTCGTTCCTCTCGCTCTCTTCAAACCCAAAACCAAAGCTGCTCCTAAAAAGGTTGAGAAGCCGAAGAGCAAGGTTGAGGATGGCATCTTTGGAACGTCTGGTGGGATTGGTTTCACAAAGGCGAATGAGCTATTCGTTGGTCGTGTTGCTATGATCGGTTTCGCTGCATCGTTGCTTGGTGAGGCGTTGACGGGAAAAGGGATATTAGCTCAGCTGAATCTGGAGACAGGGATACCGATTTACGAAGCAGAGCCATTGCTTCTCTTCTTCATCTTGTTCACTCTGTTGGGAGCCATTGGAGCTCTCGGAGACAGAGGAAAATTCGTCGACGATCCTCCCACCGGGCTCGAGAAAGCCGTCATTCCTCCCGGCAAAAACGTCCGATCTGCCCTCGGTCTCAAAGAACAAGGTCCATTGTTTGGGTTCACGAAGGCGAACGAGTTATTCGTAGGAAGATTGGCACAGTTGGGAATAGCATTTTCACTGATAGGAGAGATTATTACCGGGAAAGGAGCATTAGCTCAACTCAACATTGAGACCGGTATACCAATTCAAGATATCGAACCACTTGTCCTCTTAAACGTTGCTTTCTTCTTCTTCGCTGCCATTAATCCTGGTAATGGAAAATTCATCACCGATGATGGTGAAGAAAGCTAA SEQ ID NO: 28, Deduced amino acid sequenceof the open reading frame of Pk241MAQTMLLTSGVTAGHFLRNKSPLAQPKVHHLFLSGNSPVALPSRRQSFVPLALFKPKTKAAPKKVEKPKSKVEDGIFGTSGGIGFTKANELFVGRVAMIGFAASLLGEALTGKGILAQLNLETGIPIYEAEPLLLFFILFTLLGAIGALGDRGKFVDDPPTGLEKAVIPPGKNVRSALGLKEQGPLFGFTKANELFVGRLAQLGIAFSLIGEIITGKGALAQLNIETGIPIQDIEPLVLLNVAFFFFAAINPGNGKFITDDGEES SEQ ID NO: 29, Nucleotide sequence of theopen reading frame of Pk242ATGGGTGCAGGTGGAAGAATGCCGGTTCCTACTTCTTCCAAGAAATCGGAAACCGACACCACAAAGCGTGTGCCGTGCGAGAAACCGCCTTTCTCGGTGGGAGATCTGAAGAAAGCAATCCCGCCGCATTGTTTCAAACGCTCAATCCCTCGCTCTTTCTCCTACCTTATCAGTGACATCATTATAGCCTCATGCTTCTACTACGTCGCCACCAATTACTTCTCTCTCCTCCCTCAGCCTCTCTCTTACTTGGCTTGGCCACTCTATTGGGCCTGTCAAGGCTGTGTCCTAACTGGTATCTGGGTCATAGCCCACGAATGCGGTCACCACGCATTCAGCGACTACCAATGGCTGGATGACACAGTTGGTCTTATCTTCCATTCCTTCCTCCTCGTCCCTTACTTCTCCTGGAAGTATAGTCATCGCCGTCACCATTCCAACACTGGATCCCTCGAAAGAGATGAAGTATTTGTCCCAAAGCAGAAATCAGCAATCAAGTGGTACGGGAAATACCTCAACAACCCTCTTGGACGCATCATGATGTTAACCGTCCAGTTTGTCCTCGGGTGGCCCTTGTACTTAGCCTTTAACGTCTCTGGCAGACCGTATGACGGGTTCGCTTGCCATTTCTTCCCCAACGCTCCCATCTACAATGACCGAGAACGCCTCCAGATATACCTCTCTGATGCGGGTATTCTAGCCGTCTGTTTTGGTCTTTACCGTTACGCTGCTGCACAAGGGATGGCCTCGATGATCTGCCTCTACGGAGTACCGCTTCTGATAGTGAATGCGTTCCTCGTCTTGATCACTTACTTGCAGCACACTCATCCCTCGTTGCCTCACTACGATTCATCAGAGTGGGACTGGCTCAGGGGAGCTTTGGCTACCGTAGACAGAGACTACGGAATCTTGAACAAGGTGTTCCACAACATTACAGACACACACGTGGCTCATCACCTGTTCTCGACAATGCCGCCTTATAACGCAATGGAAGCTACAAAGGCGATAAAGCCAATTCTGGGAGACTATTACCAGTTCGATGGAACACCGTGGTATGTAGCGATGTATAGGGAGGCAAAGGAGTGTATCTATGTAGAACCGGACAGGGAAGGTGACAAGAAAGGTGTGTACTGGTACAACAATAAGTTATGA SEQ ID NO: 30,Deduced amino acid sequence of the open reading frame of Pk242MGAGGRMPVPTSSKKSETDTTKRVPCEKPPFSVGDLKKAIPPHCFKRSIPRSFSYLISDIIIASCFYYVATNYFSLLPQPLSYLAWPLYWACQGCVLTGIWVIAHECGHHAFSDYQWLDDTVGLIFHSFLLVPYFSWKYSHRRHHSNTGSLERDEVFVPKQKSAIKWYGKYLNNPLGRIMMLTVQFVLGWPLYLAFNVSGRPYDGFACHFFPNAPIYNDRERLQIYLSDAGILAVCFGLYRYAAAQGMASMICLYGVPLLIVNAFLVLITYLQHTHPSLPHYDSSEWDWLRGALATVDRDYGILNKVFHNITDTHVAHHLFSTMPPYNAMEATKAIKPILGDYYQFDGTPWYVAMYREAKECIYVEPDREGDKKGVYWYNNKL SEQ ID NO: 31, Nucleotidesequence of the open reading frame of Bn011ATGGCTTCAATAAATGAAGATGTGTCTATTGGAAACTTAGGCAGTCTCCAAACACTCCCAGACTCATTCACCTGGAAACTCACCGCTGCTGACTCCATTCTCCCTCCCTCCTCCGCCGCTGTGAAAGAGTCCATTCCGGTCATCGACCTCTCCGATCCTGACGTCACCAATTTGTTAGGAAATGCATGCAAAACGTGGGGAGCGTTTCAGATAGCCAACCACGGGGTCTCTCAAAGTCTCCTCGACGACGTTGAATCTCTCTCCAAAACCTTTTTCGATATGCCGTCAGAGAGGAAACTCGAGGCTGCTTCCTCTAATAAAGGAGTTAGTGGGTACGGAGAACCTCGAATCTCTCTTTTCTTCGAGAAGAAAATGTGGTCTGAAGGGTTGACAATCGCCGACGGCTCCTACCGCAACCAGTTCCTTACTATTTGGCCCCGTGATTACACCAAATACTGCGGAATAATCGAAGAGTACAAGGGTGAAATGGAAAAATTAGCAAGCAGACTTCTATCATGCATATTAGGATCACTTGGTGTCACCGTAGACGACATCGAATGGGCTAAGAAGACCGAGAAATCTGAATCAAAAATGGGCCAAAGCGTCATACGACTAAACCATTACCCGGTTTGTCCTGAGCCAGAAAGAGCCATGGGTCTAGCCGCTCATACCGACTCATGTCTTCTAACCATTTTGCACCAGAGCAACATGGGAGGGCTACAAGTGTTCAAAGAAGAGTCCGGTTGGGTTACGGTAGAGCCCATTCCTGGTGTTCTTGTGGTCAACATCGGCGACCTCTTTCACATTCTATCGAATGGGAAGTTTCCTAGCGTGGTTCACCGAGCAAGGGTTAACCGAACCAAGTCAAGAATATCGATAGCGTATCTGTGGGGTGGTCCAGCCGGTGAAGTGGAGATAAGTCCAATATCAAAGATAGTTGGTCCGGTTGGACCGTGTCTATACCGGCCAGTTACTTGGAGTGAATATCTCCGAATCAAATTTGAGGTTTTCGACAAGGCATTGGACGCAATTGGAGTCGTTAATCCCACCAATTGA SEQ ID NO: 32, Deduced amino acid sequence of the openreading frame of Bn011MASINEDVSIGNLGSLQTLPDSFTWKLTAADSILPPSSAAVKESIPVIDLSDPDVTNLLGNACKTWGAFQIANHGVSQSLLDDVESLSKTFFDMPSERKLEAASSNKGVSGYGEPRISLFFEKKMWSEGLTIADGSYRNQFLTIWPRDYTKYCGIIEEYKGEMEKLASRLLSCILGSLGVTVDDIEWAKKTEKSESKMGQSVIRLNHYPVCPEPERAMGLAAHTDSCLLTILHQSNMGGLQVFKEESGWVTVEPIPGVLVVNIGDLFHILSNGKFPSVVHRARVNRTKSRISIAYLWGGPAGEVEISPISKIVGPVGPCLYRPVTWSEYLRIKFEVFDKALDAIGVV NPTN SEQ IDNO: 33, Nucleotide sequence of the open reading frame of Bn077ATGGCTACATTCTCTTGTAATTCTTATGAACAAAATCACGCTCCTTTCGACCGTCACGCTAATGATACTGATATTGATGATCCTGATCATGATCATCATGATGGTGTTCAGCAAGAGGAGAGTGGATGGACAACTTATCTTGAAGATTTCTCAAATCAATACAGAACTCATCCTGAAGATAACGATCATCAAGATAAGAGTTCGTGTTCGATTCTGGACGCCTCTCCTTCTCTGGTCTCCGACGCCGCCACTGACGCATTTTCTGGCCGGAGTTTTCCAGTTAATTTTCCGGTGAAATTGAAGTTTGGGAAGGCAAGAACCAAAAAGATTTGTGAGGATGATTCTTTGGAGGATACGGCTAGCTCTCCGGTTAATAGCCCTAAGGTCAGTCAGATTGAACATATTCAGACGCCTCCTAGAAAACATGAGGACTATGTCTCTTCTAGTTTCGTTATGGGAAATATGAGTGGCATGGGGGATCATCAAATCCAAATCCAAGAAGGAGATGAACAAAAGTTGACGATGATGAGGAATCTCAGAGAAGGAAACAACAGTAACAGTAATAATATGGACTTGAGGGCTAGAGGATTATGCGTCGTCCCTATTTCCATGTTGGGTAATTTTAATGGCCGCTTCTGA SEQ ID NO: 34, Deduced amino acidsequence of the open reading frame of Bn077MATFSCNSYEQNHAPFDRHANDTDIDDPDHDHHDGVQQEESGWTTYLEDFSNQYRTHPEDNDHQDKSSCSILDASPSLVSDAATDAFSGRSFPVNFPVKLKFGKARTKKICEDDSLEDTASSPVNSPKVSQIEHIQTPPRKHEDYVSSSFVMGNMSGMGDHQIQIQEGDEQKLTMMRNLREGNNSNSNNMDLRARGLCVVPISMLGNFNGRF SEQ ID NO: 35, Nucleotidesequence of the open reading frame of Jb001ATGGCAACGGAATGCATTGCAACGGTCCCTCAAATATTCAGTGAAAACAAAACCAAAGAGGATTCTTCGATCTTCGATGCAAAGCTCCTTAATCAGCACTCACACCACATACCTCAACAGTTCGTATGGCCCGACCACGAGAAACCTTCTACGGATGTTCAACCTCTCCAAGTCCCACTCATAGACCTAGCCGGTTTCCTCTCCGGCGACTCGTGCTTGGCATCGGAGGCTACTAGACTCGTCTCAAAGGCTGCAACGAAACATGGCTTCTTCCTAATCACTAACCATGGTATCGATGAGAGCCTCTTGTCTCGTGCCTATCTGCATATGGACTCTTTCTTTAAGGCCCCGGCTTGTGAGAAGCAGAAGGCTCAGAGGAAGTGGGGTGAGAGCTCCGGTTACGCTAGTAGTTTCGTCGGGAGATTCTCCTCAAAGCTCCCGTGGAAGGAGACTCTGTCGTTTAAGTTCTCTCCCGAGGAGAAGATCCATTCCCAAACCGTTAAAGACTTTGTTTCTAAGAAAATGTGCGATGGATACGAAGATTTCGGGAAGGTTTATCAAGAATACGCGGAGGCCATGAACACTCTCTCACTAAAGATCATGGAGCTTCTTGGAATGAGTCTTGGGGTCGAGAGGAGATATTTTAAAGAGTTTTTCGAAGACAGCGATTCAATATTCCGGTTGAATTACTACCCGCAGTGCAAGCAACCGGAGCTTGCACTAGGGACAGGACCCCACTGCGACCCAACATCTCTAACCATACTTCATCAAGACCAAGTTGGCGGTCTGCAAGTTTTCGTGGACAACAAATGGCAATCCATTCCTCCTAACCCTCACGCTTTCGTGGTGAACATAGGCGACACCTTCATGGCTCTAACGAATGGAAGATACAAGAGTTGTTTGCATCGGGCGGTGGTGAACAGCGAGAGAGAAAGGAAGACGTTTGCATTCTTCCTATGTCCGAAAGGGGAAAAAGTGGTGAAGCCACCAGAAGAACTAGTAAACGGAGTGAAGTCTGGTGAAAGAAAGTATCCTGATTTTACGTGGTCTATGTTTCTCGAGTTCACACAGAAGCATTATAGGGCAGACATGAACACTCTTGACGAGTTCTCAATTTGGCTTAAGAACAGAAGAAGTTTCTAA SEQ ID NO: 36, Deducedamino acid sequence of the open reading frame of Jb001MATECIATVPQIFSENKTKEDSSIFDAKLLNQHSHHIPQQFVWPDHEKPSTDVQPLQVPLIDLAGFLSGDSCLASEATRLVSKAATKHGFFLITNHGIDESLLSRAYLHMDSFFKAPACEKQKAQRKWGESSGYASSFVGRFSSKLPWKETLSFKFSPEEKIHSQTVKDFVSKKMCDGYEDFGKVYQEYAEAMNTLSLKIMELLGMSLGVERRYFKEFFEDSDSIFRLNYYPQCKQPELALGTGPHCDPTSLTILHQDQVGGLQVFVDNKWQSIPPNPHAFVVNIGDTFMALTNGRYKSCLHRAVVNSERERKTFAFFLCPKGEKVVKPPEELVNGVKSGERKYPDFTWSMFLEFTQKHYRADMNTLDEFSIWLKNRRSF SEQ ID NO: 37, Nucleotide sequenceof the open reading frame of Jb002ATGGCGTCAGAGCAAGCAAGGAGAGAAAACAAGGTGACGGAGAGAGAAGTTCAGGTGGAGAAAGACAGAGTCCCAAAGATGACGAGTCATTTCGAGTCCATGGCCGAAAAAGGCAAAGATTCCGACACACACAGGCATCAAACAGAAGGTGGTGGGACACAGTTCGTGTCTCTCTCAGACAAGGGGAGTAACATGCCGGTTTCTGATGAAGGAGAGGGAGAGACGAAGATGAAGAGGACTCAGATGCCTCACTCCGTTGGAAAATTCGTTACTAGCAGCGATTCAGGAACAGGGAAGAAGAAGGATGAGAAAGAGGAGCATGAGAAGGCGTCGCTAGAGGATATTCATGGGTATAGAGCCAATGCTCAGCAGAAGTCAATGGATAGTATAAAAGCAGCAGAGGAAAGGTATAACAAGGCTAAGGAGAGTTTGAGCCATAGTGGACAAGAAGCTCGTGGAGGAAGAGGTGAAGAAATGGTGGGAAAAGGGCGGGACAGTGGTGTCCGTGTTTCTCACGTTGGGGCTGTTGGTGGCGGTGGTGGAGGTGAGGAAAAAGAGAGTGGTGTACATGGCTTTCATGGGGAGAAAGCACGACATGCTGAGCTTTTGGCTGCCGGAGGTGAGGAGATGAGAGAACGTGAAGGTAAAGAATCAGCAGGTGGTGTTGGTGGTCGTAGCGTAAAAGATACGGTAGCCGAGAAAGGACAGCAAGCTAAGGAAAGTGTAGGAGAAGGTGCTCAGAAAGCGGGCAGTGCTACGAGTGAGAAAGCTCAGAGAGCTTCCGAGTATGCAACAGAGAAAGGAAAAGAAGCTGGAAATATGACAGCTGAACAGGCGGCGAGAGCAAAAGACTATGCTCTGCAGAAAGCTGTTGAAGCTAAAGAGACTGCGGCGGAGAAAGCTCAGAGAGCTTCCGAGTATATGAAGGAAACAGGAAGCACAGCGGCTGAACAGGCTGCGAGAGCTAAAGATTACACTCTTCAGAAAGCTGTGGAAGCTAAAGATGTTGCAGCTGAGAAAGCTCAGAGAGCTTCAGAATACATGACAGAGACAGGAAAACAAGCCGGAAATGTTGCAGCTCAGAAAGGGCAAGAGGCAGCTTCAATGACAGCAAAAGCTAAAGATTATACTGTTCAGAAAGCCGGTGAAGCAGCTGGGTACATAAAAGAAACGACAGTGGAAGGAGGAAAAGGAGCTGCACATTATGCAGGAGTGGCAGCTGAGAAAGCCGCTGCGGTTGGGTGGACAGCGGCACATTTCACCACGGAGAAAGTGGTGCAAGGGACGAAAGCGGTTGCAGGTACAGTGGAAGGTGCTGTGGGGTACGCAGGGCATAAGGCGGTGGAAGTAGGATCTAAGGCAGTGGACTTGACTAAGGAGAAAGCTGCAGTGGCTGCTGATACGGTGGTTGGGTATACGGCGAGGAAGAAAGAGGAAGCTCAACACAGAGACCAAGAGATGCATCAGGGAGGTGAGGAAGAAAAGCAACCAGGGTTTGTCTCAGGAGCAAGGAGAGACTTTGGAGAAGAGTACGGGGAAGAAAGAGGGAGTGAGAAAGATGTCTACGGCTATGGAGCAAAAGGAATACCCGGAGAAGGGAGGGGAGATGTTGGGGAGGCAGAGTACGGAAGAGGGAGTGAGAAAGATGTCTTCGGATATGGACCAAAAGGCACGGTCGAAGAAGCAAGGAGAGACGTTGGAGAAGAATACGGAGGAGGAAGAGGCAGTGAGAGATATGTTGAAGAAGAAGGGGTTGGAGCGGGAGGGGTGCTTGGGGCAATCGGCGAGACTATAGCTGAGATTGCACAGACGACAAAGAACATAGTGATTGGTGATGCGCCTGTGAGGACACATGAGCATGGAACTACTGATCCTGACTATATGAGACGGGAACATGGACAACGTTGA SEQ ID NO: 38, Amino acid sequence of theopen reading frame of Jb002MASEQARRENKVTEREVQVEKDRVPKMTSHFESMAEKGKDSDTHRHQTEGGGTQFVSLSDKGSNMPVSDEGEGETKMKRTQMPHSVGKFVTSSDSGTGKKKDEKEEHEKASLEDIHGYRANAQQKSMDSIKAAEERYNKAKESLSHSGQEARGGRGEEMVGKGRDSGVRVSHVGAVGGGGGGEEKESGVHGFHGEKARHAELLAAGGEEMREREGKESAGGVGGRSVKDTVAEKGQQAKESVGEGAQKAGSATSEKAQRASEYATEKGKEAGNMTAEQAARAKDYALQKAVEAKETAAEKAQRASEYMKETGSTAAEQAARAKDYTLQKAVEAKDVAAEKAQRASEYMTETGKQAGNVAAQKGQEAASMTAKAKDYTVQKAGEAAGYIKETTVEGGKGAAHYAGVAAEKAAAVGWTAAHFTTEKVVQGTKAVAGTVEGAVGYAGHKAVEVGSKAVDLTKEKAAVAADTVVGYTARKKEEAQHRDQEMHQGGEEEKQPGFVSGARRDFGEEYGEERGSEKDVYGYGAKGIPGEGRGDVGEAEYGRGSEKDVFGYGPKGTVEEARRDVGEEYGGGRGSERYVEEEGVGAGGVLGAIGETIAEIAQTTKNIVIGDAPVRTHEHGTTDPDYMRREHGQR SEQ ID NO: 39, Nucleotide sequence ofthe open reading frame of Jb003ATGGCTAAGTCTTGCTATTTCAGACCAGCTCTTCTTCTTCTGTTAGTTCTTTTGGTTCATGCCGAGTCACGCGGTCGGTTCGAGCCAAAGATTCTTATGCCGACAGAGGAAGCTAACCCGGCTGACCAAGACGGAGATGGTGTCGGTACAAGATGGGCGGTTCTCGTCGCTGGTTCTTCTGGATATGGAAACTACAGACACCAGGCTGACATGTGTCACGCATATCAAATACTAAGAAAAGGAGGTTTAAAGGAAGAGAACATAGTCGTTTTGATGTATGATGATATCGCAAACCACCCACTTAATCCTCGTCCGGGTACTCTCATCAACCATCCTGACGGTGACGATGTTTACGCCGGAGTCCCTAAGGACTATACTGGTAGTAGTGTTACGGCTGCAAACTTCTACGCTGTACTCCTAGGCGACCAGAAGGCTGTTAAAGGTGGAAGCGGTAAGGTCATCGCTAGCAAGCCCAACGATCACATTTTCGTATATTATGCGGATCATGGTGGTCCCGGAGTTCTTGGGATGCCAAATACGCCTCACATATATGCAGCTGATTTTATTGAAACGCTTAAGAAGAAGCATGCTTCCGGAACATACAAAGAGATGGTTATATACGTAGAAGCGTGTGAAAGTGGGAGTATTTTCGAAGGGATAATGCCAAAGGACTTGAACATTTACGTAACAACGGCTTCAAATGCACAAGAGAGTAGTTATGGAACATATTGTCCTGGCATGAATCCGTCACCCCCATCTGAATATATCACTTGCTTAGGGGATTTATATAGTGTTGCTTGGATGGAAGATAGTGAGACTCACAATTTAAAGAAAGAGACCATAAAGCAACAATACCACACGGTGAAGATGAGGACATCAAACTACAATACCTACTCAGGTGGCTCTCATGTGATGGAATACGGTAACAATAGTATTAAGTCGGAGAAGCTTTATCTTTACCAAGGGTTTGATCCAGCCACCGTTAATCTCCCACTAAACGAATTACCGGTCAAGTCAAAAATAGGAGTCGTTAACCAACGCGATGCGGACCTTCTCTTCCTTTGGCATATGTATCGGACATCGGAAGATGGGTCAAGGAAGAAGGATGACACATTGAAGGAATTAACTGAGACAACAAGGCATAGGAAACATTTAGATGCAAGCGTCGAATTGATAGCCACAATTTTGTTTGGTCCGACGATGAATGTTCTTAACTTGGTTAGAGAACCCGGTTTGCCTTTGGTTGACGATTGGGAATGTCTTAAATCGATGGTACGTGTATTTGAAGAGCATTGTGGATCACTAACGCAATATGGGATGAAACATATGCGAGCGTTTGCAAACGTTTGTAACAACGGTGTGTCCAAAGAGCTGATGGAGGAAGCTTCTACTGCGGCATGCGGTGGTTATAGTGAGGCTCGCTACACGGTGCATCCATCAATCTTAGGCTATAGCGCCTGA SEQ ID NO: 40, Deduced aminoacid sequence of the open reading frame of Jb003MAKSCYFRPALLLLLVLLVHAESRGRFEPKILMPTEEANPADQDGDGVGTRWAVLVAGSSGYGNYRHQADMCHAYQILRKGGLKEENIVVLMYDDIANHPLNPRPGTLINHPDGDDVYAGVPKDYTGSSVTAANFYAVLLGDQKAVKGGSGKVIASKPNDHIFVYYADHGGPGVLGMPNTPHIYAADFIETLKKKHASGTYKEMVIYVEACESGSIFEGIMPKDLNIYVTTASNAQESSYGTYCPGMNPSPPSEYITCLGDLYSVAWMEDSETHNLKKETIKQQYHTVKMRTSNYNTYSGGSHVMEYGNNSIKSEKLYLYQGFDPATVNLPLNELPVKSKIGVVNQRDADLLFLWHMYRTSEDGSRKKDDTLKELTETTRHRKHLDASVELIATILFGPTMNVLNLVREPGLPLVDDWECLKSMVRVFEEHCGSLTQYGMKHMRAFANVCNNGVSKELMEEASTAACGGYSEARYTVHPSILGYSA SEQ ID NO: 41, Nucleotidesequence of the open reading frame of Jb005ATGGACGGTGCCGGAGAATCACGACTCGGTGGTGATGGTGGTGGTGATGGTTCTGTTGGAGTTCAGATCCGACAAACACGGATGCTACCGGATTTTCTCCAGAGCGTGAATCTCAAGTATGTGAAATTAGGTTACCATTACTTAATCTCAAATCTCTTGACTCTCTGTTTATTCCCTCTCGCCGTTGTTATCTCCGTCGAAGCCTCTCAGATGAACCCAGATGATCTCAAACAGCTCTGGATCCATCTACAATACAATCTGGTTAGTATCATCATCTGTTCAGCGATTCTAGTCTTCGGGTTAACGGTTTATGTTATGACCCGACCTAGACCCGTTTACTTGGTTGATTTCTCTTGTTATCTCCCACCTGATCATCTCAAAGCTCCTTACGCTCGGTTCATGGAACATTCTAGACTCACCGGAGATTTCGATGACTCTGCTCTCGAGTTTCAACGCAAGATCCTTGAGCGTTCTGGTTTAGGGGAAGACACTTATGTCCCTGAAGCTATGCATTATGTTCCACCGAGAATTTCAATGGCTGCTGCTAGAGAAGAAGCTGAACAAGTCATGTTTGGTGCTTTAGATAACCTTTTCGCTAACACTAATGTGAAACCAAAGGATATTGGAATCCTTGTTGTGAATTGTAGTCTCTTTAATCCAACTCCTTCGTTATCTGCAATGATTGTGAACAAGTATAAGCTTAGAGGTAACATTAGAAGCTACAATCTAGGCGGTATGGGTTGCAGCGCGGGAGTTATCGCTGTGGATCTTGCTAAAGACATGTTGTTGGTACATAGGAACACTTATGCGGTTGTTGTTTCTACTGAGAACATTACTCAGAATTGGTATTTTGGTAACAAGAAATCGATGTTGATACCGAACTGCTTGTTTCGAGTTGGTGGCTCTGCGGTTTTGCTATCGAACAAGTCGAGGGACAAGAGACGGTCTAAGTACAGGCTTGTACATGTAGTCAGGACTCACCGTGGAGCAGATGATAAAGCTTTCCGTTGTGTTTATCAAGAGCAGGATGATACAGGGAGAACCGGGGTTTCGTTGTCGAAAGATCTAATGGCGATTGCAGGGGAAACTCTCAAAACCAATATCACTACATTGGGTCCTCTTGTTCTACCGATAAGTGAGCAGATTCTCTTCTTTATGACTCTAGTTGTGAAGAAGCTCTTTAACGGTAAAGTGAAACCGTATATCCCGGATTTCAAACTTGCTTTCGAGCATTTCTGTATCCATGCTGGTGGAAGAGCTGTGATCGATGAGTTAGAGAAGAATCTGCAGCTTTCACCAGTTCATGTCGAGGCTTCGAGGATGACTCTTCATCGATTTGGTAACACATCTTCGAGCTCCATTTGGTATGAATTGGCTTACATTGAAGCGAAGGGAAGGATGCGAAGAGGTAATCGTGTTTGGCAAATCGCGTTCGGAAGTGGATTTAAATGTAATAGCGCGATTTGGGAAGCATTAAGGCATGTGAAACCTTCGAACAACAGTCCTTGGGAAGATTGTATTGACAAGTATCCGGTAACTTTA AGTTATTAG SEQ IDNO: 42, Deduced amino acid sequence of the open reading frame of Jb005MDGAGESRLGGDGGGDGSVGVQIRQTRMLPDFLQSVNLKYVKLGYHYLISNLLTLCLFPLAVVISVEASQMNPDDLKQLWIHLQYNLVSIIICSAILVFGLTVYVMTRPRPVYLVDFSCYLPPDHLKAPYARFMEHSRLTGDFDDSALEFQRKILERSGLGEDTYVPEAMHYVPPRISMAAAREEAEQVMFGALDNLFANTNVKPKDIGILVVNCSLFNPTPSLSAMIVNKYKLRGNIRSYNLGGMGCSAGVIAVDLAKDMLLVHRNTYAVVVSTENITQNWYFGNKKSMLIPNCLFRVGGSAVLLSNKSRDKRRSKYRLVHVVRTHRGADDKAFRCVYQEQDDTGRTGVSLSKDLMAIAGETLKTNITTLGPLVLPISEQILFFMTLVVKKLFNGKVKPYIPDFKLAFEHFCIHAGGRAVIDELEKNLQLSPVHVEASRMTLHRFGNTSSSSIWYELAYIEAKGRMRRGNRVWQIAFGSGFKCNSAIWEALRHVKPSNNSPWEDCIDKYP VTLSY SEQ IDNO: 43, Nucleotide sequence of the open reading frame of Jb007ATGTCGAGAGCTTTGTCAGTCGTTTGTGTCTTGCTCGCCATATCCTTCGTCTGTGCACGTGCTCGTCAGGTGCCGGGAGAGTCTGATGAGGGAAAGACGACGGGACATGACGATACAACAACAATGCCCATGCATGCAAAAGCAGCTGATCAGTTACCACCAAAGAGCGTCGGCGACAAAAAATGCATCGGAGGAGTTGCTGGAGTCGGTGGATTCGCCGGAGTTGGTGGTGTTGCCGGCGTGGGAGGTCTAGGGATGCCACTCATCGGTGGTCTTGGCGGGATCGGTAAGTATGGTGGCATAGGCGGTGCAGCTGGAATCGGTGGATTTCATAGTATAGGCGGTGTTGGCGGTCTAGGCGGTGTCGGAGGAGGTGTTGGCGGTCTAGGCGGTGTTGGAGGGGGTGTTGGTGGTCTAGGTGGCGTTGGCGGTCTAGGTGGAGCTGGTTTAGGCGGTGTAGGTGGTGTTGGCGGTGGTATTGGTAAAGCCGGTGGTATTGGCGGTTTAGGTGGTCTAGGCGGAGCCGGAGGTGGTTTAGGTGGAGTTGGTGGTCTCGGTAAGGCTGGTGGTATTGGTGTTGGTGGTGGTATCGGTGGTGGACACGGCGTGGTCGGTGGTGTGATCGATCCACATCCTTAA SEQ ID NO: 44, Deduced amino acidsequence of the open reading frame of Jb007MSRALSVVCVLLAISFVCARARQVPGESDEGKTTGHDDTTTMPMHAKAADQLPPKSVGDKKCIGGVAGVGGFAGVGGVAGVGGLGMPLIGGLGGIGKYGGIGGAAGIGGFHSIGGVGGLGGVGGGVGGLGGVGGGVGGLGGVGGLGGAGLGGVGGVGGGIGKAGGIGGLGGLGGAGGGLGGVGGLGKAGGIGVGGGIGGGHGVVGGVIDPHP SEQ ID NO: 45, Nucleotidesequence of the open reading frame of Jb009ATGGCAAGCAGCGACGTGAAGCTGATCGGTGCATGGGCGAGTCCCTTTGTGATGAGGCCGAGGATTGCTCTAAACCTCAAGTCTGTCCCCTACGAGTTCCTCCAAGAGACGTTTGGGTCTAAGAGCGAGTTGCTTCTTAAATCAAACCCGGTTCACAAGAAGATCCCGGTTCTGCTTCATGCTGATAAACCGGTGAGTGAGTCCAACATCATCGTTGAGTATATCGATGACACTTGGAGCTCATCTGGACCGTCCATTCTCCCTTCCGATCCTTACGATCGGGCCATGGCTCGGTTCTGGGCTGCTTACATCGACGAAAAGTGGTTTGTCGCTCTAAGAGGTTTCCTAAAAGCCGGAGGAGAAGAAGAGAAGAAAGCTGTGATAGCTCAACTAGAAGAAGGGAATGCGTTTCTGGAGAAGGCGTTCATTGATTGCAGCAAAGGAAAACCGTTCTTCAACGGTGACAACATCGGTTACCTCGACATTGCTCTCGGGTGCTTCTTGGCTTGGTTGAGAGTCACCGAGTTAGCAGTCAGCTATAAAATTCTTGATGAGGCCAAGACACCTTCTTTGTCCAAATGGGCTGAGAATTTCTGTAATGATCCCGCTGTAAAACCTGTCATGCCCGAGACTGCAAAGCTTGCTGAATTCGCAAAGAAGATCTTTCCTAAGCCGCAGGCCTAA SEQ ID NO: 46, Deduced amino acid sequence ofthe open reading frame of Jb009MASSDVKLIGAWASPFVMRPRIALNLKSVPYEFLQETFGSKSELLLKSNPVHKKIPVLLHADKPVSESNIIVEYIDDTWSSSGPSILPSDPYDRAMARFWAAYIDEKWFVALRGFLKAGGEEEKKAVIAQLEEGNAFLEKAFIDCSKGKPFFNGDNIGYLDIALGCFLAWLRVTELAVSYKILDEAKTPSLSKWAENFCNDPAVKPVMPETAKLAEFAKKIFPKPQA SEQ ID NO: 47,Nucleotide sequence of the open reading frame of Jb013ATGGCGTCTCAACAAGAGAAGAAGCAGCTGGATGAGAGGGCAAAGAAGGGCGAGACCGTCGTGCCAGGTGGTACGGGAGGCAAAAGCTTCGAAGCTCAACAGCATCTCGCTGAAGGGAGGAGCCGAGGAGGGCAAACTCGAAAGGAGCAGTTAGGAACTGAAGGATATCAGCAGATGGGACGCAAAGGTGGTCTTAGCACCGGAGACAAGCCTGGTGGGGAACACGCTGAGGAGGAAGGAGTCGAGATAGACGAATCCAAATTCAGG ACCAAGACCTAA SEQID NO: 48, Deduced amino acid sequence of the open reading frame ofJb013 MASQQEKKQLDERAKKGETVVPGGTGGKSFEAQQHLAEGRSRGGQTRKEQLGTEGYQQMGRKGGLSTGDKPGGEHAEEEGVEIDESKFRTKT SEQ ID NO: 51, Nucleotide sequenceof the open reading frame of Jb017ATGGCTCCTTCAACAAAAGTTCTCTCTTTACTTCTCTTATATGGCGTCGTGTCATTAGCCTCCGGTGATGAGTCCATCATCAACGACCATCTCCAACTTCCATCGGACGGCAAGTGGAGAACCGATGAAGAAGTGAGGTCCATCTACTTACAATGGTCCGCAGAACACGGGAAAACTAACAACAACAACAACGGTATCATCAACGACCAAGACAAAAGATTCAATATTTTCAAAGACAACTTAAGATTCATCGATCTACACAACGAAAACAACAAGAACGCTACTTACAAGCTTGGTCTCACCAAATTTACCGATCTCACTAACGATGAGTACCGCAAGTTGTACCTCGGGGCAAGAACTGAGCCCGCCCGCCGCATCGCTAAGGCCAAGAATGTCAACCAGAAATACTCAGCCGCTGTAAACGGCAAGGAGGTTCCAGAGACGGTTGATTGGAGACAGAAAGGAGCCGTTAACCCCATCAAAGACCAAGGAACTTGCGGAAGTTGTTGGGCGTTTTCGACTACTGCAGCAGTAGAAGGTATAAACAAGATCGTAACAGGAGAACTCATATCTCTATCAGAACAAGAACTTGTTGACTGCGACAAATCCTACAATCAAGGTTGCAACGGCGGTTTAATGGACTACGCTTTTCAATTCATCATGAAAAATGGTGGCTTAAACACTGAGAAAGATTATCCTTACCGTGGATTCGGCGGAAAATGCAATTCTTTCTTGAAGAATTCTAGAGTTGTGAGTATTGATGGGTACGAAGATGTTCCTACTAAAGACGAGACTGCGTTGAAGAAAGCTATTTCATACCAACCGGTTAGTGTAGCTATTGAAGCCGGTGGAAGAATTTTTCAACATTACCAATCGGGTATTTTTACCGGAAGTTGTGGTACAAATCTTGATCACGCGGTAGTTGCTGTCGGGTACGGATCAGAGAACGGTGTTGACTACTGGATTGTAAGGAACTCTTGGGGTCCACGTTGGGGTGAGGAAGGTTACATTAGAATGGAGAGAAACTTGGCAGCCTCCAAATCCGGTAAGTGTGGGATTGCGGTTGAAGCCTCGTACCCGGTTAAGTACAGCCCAAACCCGGTTCGTGGAAATACTATCAGCAGTGTTTGA SEQ ID NO: 52, Amino acidsequence of the open reading frame of Jb017MAPSTKVLSLLLLYGVVSLASGDESIINDHLQLPSDGKWRTDEEVRSIYLQWSAEHGKTNNNNNGIINDQDKRFNIFKDNLRFIDLHNENNKNATYKLGLTKFTDLTNDEYRKLYLGARTEPARRIAKAKNVNQKYSAAVNGKEVPETVDWRQKGAVNPIKDQGTCGSCWAFSTTAAVEGINKIVTGELISLSEQELVDCDKSYNQGCNGGLMDYAFQFIMKNGGLNTEKDYPYRGFGGKCNSFLKNSRVVSIDGYEDVPTKDETALKKAISYQPVSVAIEAGGRIFQHYQSGIFTGSCGTNLDHAVVAVGYGSENGVDYWIVRNSWGPRWGEEGYIRMERNLAASKSGKCGIAVEASYPVKYSPNPVRGNTISSV SEQ ID NO: 53, Nucleotide sequenceof the open reading frame of Jb024ATGCGGTGCTTTCCACCTCCCTTATGGTGCACCTCCTTGGTCGTTTTCTTGTCGGTTACCGGAGCCCTAGCCGCCGATCCCTACGTCTTCTTCGATTGGACTGTCTCTTACCTCTCTGCTTCTCCTCTCGGCACTCGTCAACAGGTAATTGGGATAAATGGGCAATTTCCTGGTCCGATTCTAAACGTAACTACGAATTGGAATGTTGTTATGAATGTGAAGAATAATCTTGATGAGCCATTGCTTCTTACATGGAATGGAATCCAACATAGGAAAAACTCATGGCAAGATGGTGTTTTGGGAACTAATTGTCCAATTCCTTCTGGTTGGAATTGGACTTATGAGTTTCAAGTTAAAGATCAGATTGGTAGTTTCTTTTATTTTCCTTCTACAAATTTTCAAAGAGCTTCTGGTGGTTATGGAGGGATTATTGTCAATAATCGCGCTATCATTCCGGTTCCTTTCGCTCTTCCTGATGGTGATGTTACTCTCTTTATCAGTGATTGGTATACTAAGAGCCATAAGAAGCTGAGGAAGGATGTTGAGAGTAAGAACGGCCTTCGACCTCCGGATGGTATTGTCATCAATGGATTTGGACCTTTTGCTTCTAATGGTAGTCCTTTTGGGACCATAAACGTTGAACCAGGACGAACATATCGTTTTCGTGTTCACAATAGTGGCATTGCGACCAGCTTGAATTTCAGAATACAGAATCATAACCTGCTTCTTGTTGAGACAGAAGGGTCATACACAATTCAGCAGAATTATACGAATATGGATATACATGTGGGTCAATCTTTCTCATTTCTGGTCACTATGGATCAGTCTGGTAGTAATGACTACTACATTGTTGCCAGCCCAAGGTTTGCTACATCCATCAAAGCTAGTGGAGTCGCTGTCTTGCGCTACTCTAATTCCCAAGGACCCGCTTCAGGTCCACTCCCTGATCCTCCTATTGAGTTGGACACATTTTTCTCAATGAACCAAGCACGATCCTTAAGGTTGAATTTGTCATCTGGAGCTGCCCGTCCAAACCCGCAGGGATCTTTCAAATATGGCCAGATTACAGTAACTGATGTGTATGTGATTGTCAACCGACCACCAGAGATGATAGAGGGACGATTGCGTGCAACTCTTAATGGTATATCATACTTACCTCCTGCAACACCCCTAAAGCTTGCTCAGCAATACAACATCTCAGGGGTATACAAGTTGGATTTCCCAAAAAGGCCAATGAATAGGCACCCCAGGGTTGATACCTCAGTCATAAACGGCACGTTCAAGGGATTCGTGGAAATCATATTTCAAAATAGTGACACCACTGTTAAGAGCTACCACTTGGATGGTTATGCATTTTTTGTTGTTGGGATGGACTTTGGTCTGTGGACAGAAAATAGCAGAAGCACATACAACAAGGGTGATGCAGTTGCTCGATCTACTACGCAGGTGTTTCCTGGTGCATGGACGGCCGTCTTGGTTTCTTTGGACAATGCTGGCATGTGGAACCTTCGAATAGACAATCTAGCCTCATGGTATCTTGGCCAAGAACTATACTTGAGTGTGGTTAATCCAGAGATTGACATTGACTCATCTGAGAATTCCGTTCCTAAAAACTCTATATATTGTGGTCGGCTCTCACCATTACAAAAGTAA SEQ ID NO: 54,Deduced amino acid sequence of the open reading frame of Jb024MRCFPPPLWCTSLVVFLSVTGALAADPYVFFDWTVSYLSASPLGTRQQVIGINGQFPGPILNVTTNWNVVMNVKNNLDEPLLLTWNGIQHRKNSWQDGVLGTNCPIPSGWNWTYEFQVKDQIGSFFYFPSTNFQRASGGYGGIIVNNRAIIPVPFALPDGDVTLFISDWYTKSHKKLRKDVESKNGLRPPDGIVINGFGPFASNGSPFGTINVEPGRTYRFRVHNSGIATSLNFRIQNHNLLLVETEGSYTIQQNYTNMDIHVGQSFSFLVTMDQSGSNDYYIVASPRFATSIKASGVAVLRYSNSQGPASGPLPDPPIELDTFFSMNQARSLRLNLSSGAARPNPQGSFKYGQITVTDVYVIVNRPPEMIEGRLRATLNGISYLPPATPLKLAQQYNISGVYKLDFPKRPMNRHPRVDTSVINGTFKGFVEIIFQNSDTTVKSYHLDGYAFFVVGMDFGLWTENSRSTYNKGDAVARSTTQVFPGAWTAVLVSLDNAGMWNLRIDNLASWYLGQELYLSVVNPEIDIDSSENSVPKNSIYCGRLSPLQK SEQ ID NO: 55, Nucleotide sequenceof the open reading frame of Jb027ATGCTTCTAATTCTAGCGATTTGGTCACCAATTTCACACTCGCTTCACTTCGATCTACACTCAGGTCGCACAAAGTGTATCGCCGAAGACATCAAAAGCAATTCAATGACTGTTGGTAAATACAACATCGATAATCCTCACGAAGGTCAAGCTTTACCACAAACTCACAAAATTTCCGTCAAGGTGACGTCTAATTCCGGTAACAATTACCATCACGCGGAACAAGTAGATTCAGGACAATTCGCATTCTCGGCTGTTGAAGCAGGTGATTACATGGCTTGTTTCACTGCTGTTGATCATAAGCCTGAGGTTTCGTTGAGTATTGACTTTGAGTGGAAGACTGGTGTTCAATCTAAAAGCTGGGCTAATGTTGCTAAGAAGAGTCAAGTCGAAGTTATGGAATTTGAAGTAAAGAGTCTTCTTGATACTGTTAACTCGATTCATGAAGAGATGTATTATCTTAGAGATAGGGAAGAAGAGATGCAAGACTTGAACCGGTCCACTAACACAAAAATGGCGTGGTTGAGTGTTCTCTCGTTTTTCGTCTGCATAGGAGTTGCAGGGATGCAGTTTTTGCACTTGAAGACGTTTTTCGAGAAGAAGA AGGTTATCTGA SEQID NO: 56, Deduced amino acid sequence of the open reading frame ofJb027 MLLILAIWSPISHSLHFDLHSGRTKCIAEDIKSNSMTVGKYNIDNPHEGQALPQTHKISVKVTSNSGNNYHHAEQVDSGQFAFSAVEAGDYMACFTAVDHKPEVSLSIDFEWKTGVQSKSWANVAKKSQVEVMEFEVKSLLDTVNSIHEEMYYLRDREEEMQDLNRSTNTKMAWLSVLSFFVCIGVAGMQFLHLKTFFEKKKVI SEQ ID NO: 57, Nucleotide sequenceof the open reading frame of OO-1ATGGCACATGCCACGTTTACGTCGGAAGGGCAGAATATGGAGTCGTTTCGACTCTTGAGTGGCCACAAAATCCCAGCCGTTGGACTCGGCACGTGGCGATCTGGGTCTCAAGCCGCCCACGCCGTTGTCACTGCAATCGTCGAGGGTGGCTATAGGCACATAGATACAGCTTGGGAGTATGGTGATCAGAGAGAGGTCGGTCAAGGAATAAAGAGGGCGATGCACGCTGGCCTTGAAAGGAGGGACCTCTTTGTGACCTCGAAGCTTTGGTGCACTGAGTTATCTCCTGAGAGAGTGCGTCCTGCTCTGCAAAACACCCTTAAAGAGCTTCAATTAGAGTACCTTGATCTCTACTTGATTCACTGGCCTATCCGGCTAAGAGAAGGAGCCAGTAAGCCACCAAAGGCAGGGGACGTTCTTGACTTTGACATGGAAGGAGTTTGGAGAGAAATGGAGAATCTTTCCAAGGACAGTCTCGTCAGGAATATCGGTGTCTGTAACTTTACAGTCACTAAGCTCAATAAGCTGCTAGGATTTGCTGAACTGATCCCTGCCGTTTGCCAGATGGAAATGCATCCTGGTTGGAGAAACGATAGGATACTCGAATTCTGCAAGAAGAATGAGATCCATGTTACTGCCTATTCTCCATTGGGATCTCAAGAAGGCGGGAGAGATCTGATACACGATCAGACGGTGGATAGGATAGCGAAGAAGCTGAATAAGACACCGGGACAGATTCTAGTGAAATGGGGTTTGCAGAGAGGAACAAGTGTCATCCCTAAGTCATTGAATCCAGAGAGGATCAAAGAGAACATCAAAGTGTTTGATTGGGTGATCCCTGAACAAGACTTCCAAGCTCTCAACAGCATCACTGACCAGAAACGAGTGATAGACGGTGAGGATCTTTTCGTCAACAAGACCGAAGGTCCATTCCGTAGTGTGGCTGATCTATGGGACCATGAAGACTAA SEQ ID NO: 58, Deduced aminoacid sequence of the open reading frame of OO-1MAHATFTSEGQNMESFRLLSGHKIPAVGLGTWRSGSQAAHAVVTAIVEGGYRHIDTAWEYGDQREVGQGIKRAMHAGLERRDLFVTSKLWCTELSPERVRPALQNTLKELQLEYLDLYLIHWPIRLREGASKPPKAGDVLDFDMEGVWREMENLSKDSLVRNIGVCNFTVTKLNKLLGFAELIPAVCQMEMHPGWRNDRILEFCKKNEIHVTAYSPLGSQEGGRDLIHDQTVDRIAKKLNKTPGQILVKWGLQRGTSVIPKSLNPERIKENIKVFDWVIPEQDFQALNSITDQKRVIDGEDLFVNKTEGPFRSVADLWDHED SEQ ID NO: 59, Nucleotidesequence of the open reading frame of OO-2ATGGCGTCTGAGAAACAAAAACAACATGCACAACCTGGCAAAGAACATGTCATGGAATCAAGCCCACAATTCTCTAGCTCAGATTACCAACCTTCCAACAAGCTTCGTGGTAAGGTGGCGTTGATAACTGGTGGAGACTCTGGGATTGGTCGAGCCGTGGGATACTGTTTTGCATCCGAAGGAGCTACTGTGGCTTTCACTTACGTGAAGGGTCAAGAAGAAAAAGATGCACAAGAGACCCTACAAATGTTGAAGGAGGTCAAAACCTCGGACTCCAAGGAACCTATCGCCATTCCAACGGATTTAGGATTTGACGAAAACTGCAAAAGGGTCGTTGATGAGGTCGTTAATGCTTTTGGCCGCATCGATGTTTTGATCAATAACGCAGCAGAGCAGTACGAGAGCAGCACAATCGAAGAGATTGATGAGCCTAGGCTTGAGCGAGTCTTCCGTACAAACATCTTTTCTTACTTCTTTCTCACAAGGCATGCGTTGAAGCATATGAAGGAAGGAAGCAGCATTATCAACACCACTTCGGTGAATGCCTACAAGGGAAACGCTTCACTTCTCGACTACACCGCTACAAAAGGAGCGATTGTGGCGTTTACTCGAGGACTTGCACTTCAGCTAGCTGAGAAAGGAATCCGTGTCAATGGTGTGGCTCCTGGTCCAATATGGACACCCCTTATCCCAGCATCATTCAATGAGGAGAAGATTAAGAATTTTGGGTCTGAGGTTCCGATGAAAAGAGCGGGTCAGCCAATTGAAGTGGCACCATCCTATGTTTTCTTGGCGTGTAACCACTGCTCTTCTTACTTCACTGGTCAAGTTCTTCACCCTAATGGAGGAGCTGTGGTAAATGCGTAA SEQ ID NO: 60, Deducedamino acid sequence of the open reading frame of OO-2MASEKQKQHAQPGKEHVMESSPQFSSSDYQPSNKLRGKVALITGGDSGIGRAVGYCFASEGATVAFTYVKGQEEKDAQETLQMLKEVKTSDSKEPIAIPTDLGFDENCKRVVDEVVNAFGRIDVLINNAAEQYESSTIEEIDEPRLERVFRTNIFSYFFLTRHALKHMKEGSSIINTTSVNAYKGNASLLDYTATKGAIVAFTRGLALQLAEKGIRVNGVAPGPIWTPLIPASFNEEKIKNFGSEVPMKRAGQPIEVAPSYVFLACNHCSSYFTGQVLHPNGGAVVNA SEQ ID NO: 61,Nucleotide sequence of the open reading frame of OO-3ATGGATTCAACGAAGCTTAGTGAGCTAAAGGTCTTCATCGATCAATGCAAGTCTGACCCTTCCCTTCTCACTACTCCTTCACTCTCCTTCTTCCGTGACTATCTCGAGAGTCTTGGTGCTAAGATACCTACTGGTGTCCATGAAGAAGACAAAGACACTAAGCCGAGGAGTTTCGTAGTGGAAGAGAGTGATGATGATATGGATGAAACTGAAGAAGTAAAACCGAAAGTGGAGGAAGAAGAAGAAGAGGATGAGATTGTTGAATCTGATGTAGAGCTTGAAGGAGACACTGTTGAGCCTGATAATGATCCTCCTCAGAAGATGGGGGATTCATCAGTGGAGGTGACTGATGAGAATCGTGAAGCTGCTCAAGAAGCTAAGGGCAAAGCCATGGAGGCCCTTTCTGAAGGAAACTTTGATGAAGCAATTGAGCATTTAACTCGGGCAATAACGTTGAACCCGACTTCAGCTATTATGTATGGAAACAGAGCTAGTGTCTACATTAAGTTGAAGAAGCCAAACGCTGCTATTCGAGATGCAAACGCAGCATTGGAGATTAACCCTGATTCTGCCAAGGGATACAAGTCACGAGGTATGGCTCGTGCCATGCTTGGAGAATGGGCAGAGGCTGCAAAAGACCTTCACCTTGCATCTACGATAGACTATGATGAGGAAATTAGTGCTGTTCTCAAAAAGGTTGAACCTAATGCACATAAGCTTGAGGAGCACCGTAGAAAGTATGACAGATTACGTAAGGAAAGAGAGGACAAAAAGGCTGAACGGGATAGATTACGTCGCCGTGCTGAAGCACAGGCTGCCTATGATAAAGCTAAGAAAGAAGAACAGTCATCATCTAGCAGACCATCAGGAGGCGGTTTCCCAGGAGGTATGCCCGGTGGTTTCCCAGGAGGTATGCCCGGTGGATTCCCAGGAGGAATGGGAGGCATGCCCGGCGGATTCCCGGGAGGAATGGGTGGTATGGGCGGTATGCCCGGTGGATTCCCAGGAGGAATGGGCGGTGGTATGCCTGCAGGAATGGGCGGTGGTATGCCCGGAATGGGCGGTGGTATGCCTGCTGGAATGGGTGGTGGCGGTATGCCAGGTGCAGGCGGTGGTATGCCTGGTGGTGGCGGTATGCCTGGTGGTATGGACTTCAGCAAAATATTGAATGATCCTGAGCTAATGACGGCATTTAGCGACCCTGAAGTCATGGCTGCTCTTCAAGATGTGATGAAGAACCCTGCGAATCTAGCGAAGCATCAGGCGAATCCGAAGGTGGCTCCCGTGATTGCAAAGATGATGGGCAAATTTGCAGGACCTCAGTAA SEQ ID NO: 62, Deduced amino acid sequence of theopen reading frame of OO-3MDSTKLSELKVFIDQCKSDPSLLTTPSLSFFRDYLESLGAKIPTGVHEEDKDTKPRSFVVEESDDDMDETEEVKPKVEEEEEEDEIVESDVELEGDTVEPDNDPPQKMGDSSVEVTDENREAAQEAKGKAMEALSEGNFDEAIEHLTRAITLNPTSAIMYGNRASVYIKLKKPNAAIRDANAALEINPDSAKGYKSRGMARAMLGEWAEAAKDLHLASTIDYDEEISAVLKKVEPNAHKLEEHRRKYDRLRKEREDKKAERDRLRRRAEAQAAYDKAKKEEQSSSSRPSGGGFPGGMPGGFPGGMPGGFPGGMGGMPGGFPGGMGGMGGMPGGFPGGMGGGMPAGMGGGMPGMGGGMPAGMGGGGMPGAGGGMPGGGGMPGGMDFSKILNDPELMTAFSDPEVMAALQDVMKNPANLAKHQANPKVAPVIAKMMGKFAGPQ SEQ ID NO: 63,Nucleotide sequence of the open reading frame of OO-4ATGAAGGTTCACGAGACAAGATCTCACGCTCACATGTCTGGAGACGAACAAAAGAAGGGAAATTTGCGGAAGCACAAAGCAGAAGGGAAACTTCCAGAATCTGAACAGTCTCAGAAGAAGGCAAAGCCTGAAAACGATGACGGACGTTCTGTCAACGGCGCCGGAGATGCTGCTTCAGAGTACAATGAGTTCTGCAAAGCGGTTGAGGAGAATCTGTCCATTGATCAGATTAAAGAAGTTCTCGAAATCAACGGCCAAGATTGTTCTGCTCCAGAAGAGACCTTGCTAGCTCAATGTCAAGATTTGCTGTTCTATGGGGCATTAGCTAAATGTCCTTTATGCGGAGGAACTTTAATTTGCGACAATGAAAAGAGATTTGTATGTGGAGGTGAGATAAGTGAGTGGTGCAGTTGCGTGTTTAGTACGAAAGATCCTCCTAGAAAGGAAGAGCCAGTTAAAATCCCTGATTCTGTCATGAACTCTGCTATATCTGACTTGATCAAGAAACACCAGGACCCTAAAAGCCGACCTAAAAGAGAGTTAGGCTCTGCTGATAAACCCTTTGTGGGAATGATGATCTCTCTCATGGGACGTCTCACGAGAACACATCAATATTGGAAGAAAAAGATCGAGAGAAACGGTGGGAAAGTCTCCAATACTGTTCAAGGCGTAACATGTTTGGTGGTTTCGCCAGCTGAAAGAGAACGAGGTGGTACGTCAAAGATGGTGGAGGCAATGGAACAAGGTCTACCGGTTGTGAGCGAAGCATGGTTGATCGACAGCGTGGAGAAGCATGAAGCTCAGCCACTTGAAGCTTATGACGTGGTCAGTGATCTTTCAGTGGAAGGGAAAGGAATTCCATGGGATAAGCAAGATCCTAGTGAGGAGGCAATTGAATCCTTTTCTGCTGAGCTCAAAATGTATGGGAAAAGAGGAGTGTACATGGACACAAAACTTCAGGAGAGAGGAGGAAAGATCTTCGAGAAAGATGGACTCTTGTATAACTGTGCCTTCTCGATATGCGATTTGGGAAAAGGGCGTAATGAGTATTGTATTATGCAGCTAGTCACGGTACCCGATAGTAACCTGAACATGTACTTCAAGAGAGGGAAAGTAGGAGATGACCCTAATGCCGAAGAGAGGCTCGAGGAATGGGAGGACGAAGAAGCTGCGATCAAAGAGTTTGCAAGGCTTTTTGAGGAGATAGCAGGGAATGAGTTTGAGCCATGGGAACGTGAGAAGAAGATTCAAAAGAAGCCTCATAAGTTTTTCCCAATTGATATGGATGATGGAATCGAAGTAAGGAGTGGGGCTCTTGGTCTAAGGCAGCTTGGCATTGCTTCTGCTCATTGCAAGCTTGATTCGTTTGTTGCAAACTTCATTAAAGTTCTGTGTGGTCAAGAGATTTACAATTACGCGTTGATGGAGCTTGGATTGGATCCGCCCGATCTACCTATGGGAATGCTAACTGATATCCACTTGAAACGATGCGAAGAGGTATTACTCGAGTTTGTTGAGAAGGTCAAAACAACAAAAGAGACAGGTCAGAAAGCTGAAGCAATGTGGGCAGACTTCAGCTCACGATGGTTCTCTTTGATGCACAGCACTAGGCCGATGCGATTACACGATGTCAATGAACTTGCAGACCATGCGGCCTCTGCTTTTGAGACGGTGAGGGACATAAACACAGCATCTCGTTTGATAGGGGACATGCGAGGAGACACACTCGATGATCCGTTGTCTGATAGGTACAAAAAACTTGGCTGCAAGATATCTGTGGTAGACAAAGAGTCTGAAGATTACAAGATGGTTGTGAAGTATCTCGAGACTACTTATGAGCCTGTGAAAGTCTCTGATGTTGAGTACGGTGTGTCAGTGCAGAATGTTTTTGCGGTTGAGTCAGATGCAATTCCTTCATTAGATGATATCAAGAAGTTACCAAATAAGGTCCTTTTATGGTGTGGGTCTCGGAGCTCAAATCTATTGAGACATATCTACAAAGGGTTCTTACCTGCTGTATGCTCTCTTCCGGTTCCTGGTTATATGTTTGGGAGAGCGATAGTGTGTTCAGATGCAGCTGCAGAAGCAGCAAGGTATGGTTTTACGGCTGTGGATAGACCAGAAGGGTTTCTTGTATTAGCCGTAGCATCACTTGGTGAGGAAGTTACAGAATTTACAAGTCCACCAGAGGATACGAAGACGTTGGAAGATAAAAAGATTGGAGTGAAAGGATTAGGGAGGAAGAAAACTGAAGAGTCGGAGCATTTCATGTGGAGAGATGACATAAAAGTTCCTTGTGGACGGTTGGTTCCATCGGAACATAAGGACAGTCCACTTGAGTACAACGAGTACGCGGTTTATGATCCGAAACAGACAAGTATAAGGTTCTTGGTGGAAGTGAAGTACGAGGAGAAGGGAACTGAGATAGTCGATGTCGAACCAGAGTAG SEQ ID NO: 64,Deduced amino acid sequence of the open reading frame of OO-4MKVHETRSHAHMSGDEQKKGNLRKHKAEGKLPESEQSQKKAKPENDDGRSVNGAGDAASEYNEFCKAVEENLSIDQIKEVLEINGQDCSAPEETLLAQCQDLLFYGALAKCPLCGGTLICDNEKRFVCGGEISEWCSCVFSTKDPPRKEEPVKIPDSVMNSAISDLIKKHQDPKSRPKRELGSADKPFVGMMISLMGRLTRTHQYWKKKIERNGGKVSNTVQGVTCLVVSPAERERGGTSKMVEAMEQGLPVVSEAWLIDSVEKHEAQPLEAYDVVSDLSVEGKGIPWDKQDPSEEAIESFSAELKMYGKRGVYMDTKLQERGGKIFEKDGLLYNCAFSICDLGKGRNEYCIMQLVTVPDSNLNMYFKRGKVGDDPNAEERLEEWEDEEAAIKEFARLFEEIAGNEFEPWEREKKIQKKPHKFFPIDMDDGIEVRSGALGLRQLGIASAHCKLDSFVANFIKVLCGQEIYNYALMELGLDPPDLPMGMLTDIHLKRCEEVLLEFVEKVKTTKETGQKAEAMWADFSSRWFSLMHSTRPMRLHDVNELADHAASAFETVRDINTASRLIGDMRGDTLDDPLSDRYKKLGCKISVVDKESEDYKMVVKYLETTYEPVKVSDVEYGVSVQNVFAVESDAIPSLDDIKKLPNKVLLWCGSRSSNLLRHIYKGFLPAVCSLPVPGYMFGRAIVCSDAAAEAARYGFTAVDRPEGFLVLAVASLGEEVTEFTSPPEDTKTLEDKKIGVKGLGRKKTEESEHFMWRDDIKVPCGRLVPSEHKDSPLEYNEYAVYDPKQTSIRFLVEVKYEEKGTEIVDVEPE SEQ ID NO: 65, Nucleotide sequence of the openreading frame of OO-5ATGTCTACCCCAGCTGAATCTTCAGACTCGAAATCGAAGAAAGATTTCAGTACTGCTATTCTCGAGAGGAAGAAGTCTCCGAACCGTCTCGTCGTCGATGAGGCTATCAACGATGATAACTCCGTCGTCTCTCTTCACCCTGCAACCATGGAGAAGCTTCAGCTCTTCCGTGGTGATACCATTCTCATCAAGGGTAAGAAGAGGAAGGACACTGTCTGCATTGCTCTTGCTGATGAGACATGTGAGGAGCCAAAGATCAGAATGAATAAAGTAGTCAGATCTAACTTGAGGGTTAGACTGGGAGATGTTATATCTGTTCACCAATGCCCAGACGTCAAGTACGGAAAGCGTGTTCACATCCTGCCTGTTGATGATACTGTTGAAGGAGTGACTGGAAACCTATTTGATGCTTACCTGAAACCTTATTTCCTTGAGGCATACCGTCCAGTGAGGAAGGGTGATCTCTTCCTAGTCAGAGGAGGAATGAGGAGTGTGGAGTTCAAAGTTATAGAGACAGATCCTGCTGAGTACTGCGTGGTTGCTCCAGACACAGAGATTTTCTGTGAGGGTGAGCCTGTGAAGAGAGAGGATGAAGAAAGGCTAGATGATGTAGGTTATGATGATGTTGGTGGTGTCAGGAAACAGATGGCTCAGATTAGGGAACTTGTTGAACTTCCCTTGAGGCATCCACAGCTATTCAAGTCGATTGGTGTTAAGCCACCGAAGGGAATTCTTCTTTATGGACCACCTGGGTCTGGAAAGACTTTGATCGCTCGTGCTGTGGCTAATGAAACGGGTGCCTTTTTCTTCTGTATCAACGGACCTGAGATCATGTCCAAATTGGCTGGTGAGAGTGAGAGCAACCTCAGGAAAGCATTCGAGGAGGCTGAGAAAAATGCGCCTTCAATCATATTCATTGATGAGATCGACTCTATTGCACCGAAAAGAGAGAAGACTAATGGAGAGGTTGAGAGGAGGATTGTCTCTCAGCTCCTTACGCTAATGGATGGACTGAAATCTCGTGCTCATGTTATCGTCATGGGAGCAACCAATCGCCCCAACAGTATCGACCCAGCTTTGAGAAGGTTTGGAAGATTTGACAGGGAGATCGATATTGGAGTTCCTGACGAAATTGGACGTCTTGAAGTTCTGAGGATCCATACAAAGAACATGAAGCTGGCTGAAGATGTGGATCTCGAAAGGATCTCAAAGGACACACACGGTTACGTCGGTGCTGATCTTGCAGCTTTGTGCACAGAGGCCGCCCTGCAATGCATCAGGGAGAAGATGGATGTGATTGATCTGGAAGATGACTCCATAGACGCTGAAATCCTCAATTCCATGGCAGTCACTAATGAACATTTCCACACTGCTCTCGGGAACAGCAACCCATCTGCACTTCGTGAAACTGTTGTGGAGGTTCCCAACGTCTCTTGGAATGATATTGGAGGTCTTGAGAATGTCAAGAGAGAGCTCCAGGAGACTGTTCAATACCCAGTCGAGCACCCAGAGAAGTTTGAGAAATTCGGGATGTCTCCATCAAAGGGAGTCCTTTTCTACGGTCCTCCTGGATGTGGGAAAACCCTTTTGGCCAAAGCTATTGCCAACGAGTGCCAAGCTAATTTCATCAGTGTCAAGGGTCCCGAGCTTCTGACAATGTGGTTTGGAGAGAGTGAAGCAAATGTTCGTGAAATCTTCGACAAGGCCCGTCAATCCGCTCCATGTGTTCTTTTCTTTGATGAGCTCGACTCCATTGCAACTCAGAGAGGAGGTGGAAGTGGTGGCGATGGAGGTGGTGCTGCGGACAGAGTCTTGAACCAGCTTTTGACTGAGATGGACGGAATGAATGCCAAGAAAACCGTCTTCATCATCGGAGCTACCAACAGACCTGACATTATCGATTCAGCTCTTCTCCGTCCTGGAAGGCTTGACCAGCTCATTTACATTCCACTACCAGATGAGGATTCCCGTCTCAATATCTTCAAGGCCGCCTTGAGGAAATCTCCTATTGCTAAAGATGTAGACATCGGTGCACTTGCTAAATACACTCAGGGTTTCAGTGGTGCTGATATCACTGAGATTTGCCAGAGAGCTTGCAAGTACGCCATCAGAGAAAACATTGAGAAGGACATTGAAAAGGAGAAGAGGAGGAGCGAGAACCCAGAGGCAATGGAGGAAGATGGAGTGGATGAAGTATCAGAGATCAAAGCTGCACACTTTGAGGAGTCGATGAAGTATGCGCGTAGGAGTGTGAGTGATGCAGACATCAGGAAGTACCAAGCCTTTGCTCAGACGTTGCAGCAGTCTAGAGGGTTCGGTTCTGAGTTCAGGTTCGAGAATTCTGCTGGTTCAGGTGCCACCACTGGAGTCGCAGATCCGTTTGCCACGTCTGCAGCCGCTGCTGGGGACGATGATGATCTCTACAATTAG SEQ ID NO: 66, Deduced amino acid sequence ofthe open reading frame of OO-5MSTPAESSDSKSKKDFSTAILERKKSPNRLVVDEAINDDNSVVSLHPATMEKLQLFRGDTILIKGKKRKDTVCIALADETCEEPKIRMNKVVRSNLRVRLGDVISVHQCPDVKYGKRVHILPVDDTVEGVTGNLFDAYLKPYFLEAYRPVRKGDLFLVRGGMRSVEFKVIETDPAEYCVVAPDTEIFCEGEPVKREDEERLDDVGYDDVGGVRKQMAQIRELVELPLRHPQLFKSIGVKPPKGILLYGPPGSGKTLIARAVANETGAFFFCINGPEIMSKLAGESESNLRKAFEEAEKNAPSIIFIDEIDSIAPKREKTNGEVERRIVSQLLTLMDGLKSRAHVIVMGATNRPNSIDPALRRFGRFDREIDIGVPDEIGRLEVLRIHTKNMKLAEDVDLERISKDTHGYVGADLAALCTEAALQCIREKMDVIDLEDDSIDAEILNSMAVTNEHFHTALGNSNPSALRETVVEVPNVSWNDIGGLENVKRELQETVQYPVEHPEKFEKFGMSPSKGVLFYGPPGCGKTLLAKAIANECQANFISVKGPELLTMWFGESEANVREIFDKARQSAPCVLFFDELDSIATQRGGGSGGDGGGAADRVLNQLLTEMDGMNAKKTVFIIGATNRPDIIDSALLRPGRLDQLIYIPLPDEDSRLNIFKAALRKSPIAKDVDIGALAKYTQGFSGADITEICQRACKYAIRENIEKDIEKEKRRSENPEAMEEDGVDEVSEIKAAHFEESMKYARRSVSDADIRKYQAFAQTLQQSRGFGSEFRFENSAGSGATTGVADPFATSAAAAGDDDD LYN SEQ ID NO:67, Nucleotide sequence of the open reading frame of OO-6ATGGACAAATCTAGTACCATGCTTGTTCACTATGACAAAGGGACTCCAGCAGTTGCTAATGAGATTAAAGAAGCTCTCGAAGGAAATGATGTTGAAGCTAAAGTTGATGCCATGAAGAAGGCAATTATGCTTTTGCTGAATGGTGAAACCATTCCTCAGCTTTTCATTACCATTATAAGATATGTGCTGCCTTCTGAAGACCACACCATCCAAAAGCTTCTGTTGCTGTACCTGGAGCTGATTGAAAAGACAGATTCGAAGGGGAAGGTGTTGCCTGAAATGATTTTGATATGCCAGAATCTTCGTAATAACCTTCAGCATCCGAATGAGTACATCCGTGGAGTGACACTGAGGTTTCTCTGTCGGATGAAGGAGACTGAAATAGTGGAACCTTTGACTCCATCAGTGTTACAAAATCTGGAGCATCGCCATCCATTTGTTCGCAGGAATGCAATTCTGGCAATCATGTCGATATATAAACTTCCACATGGCGACCAACTCTTCGTGGATGCACCTGAAATGATCGAGAAAGTTCTATCAACAGAACAAGATCCTTCTGCCAAGAGAAATGCATTTCTAATGCTCTTTACCTGTGCCGAAGAACGTGCAGTGAATTATCTTCTGAGCAATGTTGACAAGGTTTCAGACTGGAATGAATCACTTCAGATGGTGGTGCTGGAGCTGATTCGAAGTGTGTGTAAGACTAAACCAGCGGAGAAGGGAAAATATATTAAAATTATTATTTCTCTGTTAAGTGCTACTTCTTCTGCAGTTATCTATGAATGTGCTGGGACACTTGTTTCTCTCTCATCTGCCCCTACTGCTATTCGAGCTGCTGCCAACACCTACTGCCAACTTCTTCTTTCTCAGAGTGACAACAATGTGAAGCTTATCTTGCTCGATCGGTTGTATGAGCTTAAGACATTGCACAGAGATATCATGGTTGAGCTGATAATCGATGTGCTCAGAGCACTCTCAAGCCCAAACCTTGATATCCGCAGGAAGACACTTGACATTGCCCTTGACTTGATTACCCATCATAATATTAATGAAGTCGTTCAAATGTTGAAGAAAGAAGTTGTGAAGACACAGAGTGGAGAACTTGAGAAGAATGGAGAGTACAGGCAAATGCTTATTCAAGCCATCCATGCTTGTGCAGTTAAGTTCCCCGAAGTTGCAAGCACAGTGGTCCATCTTCTGATGGATTTCCTGGGAGATAGCAACGTGGCTTCAGCTCTTGACGTGGTTGTTTTCGTTAGAGAGATAATAGAAACAAATCCCAAGTTGAGAGTTTCAATCATCACCAGGTTGTTGGACACGTTCTATCAGATCCGTGCAGGAAAGGTCTGCCCTTGTGCACTTTGGATCATTGGTGAGTATTGCCTATCACTTTCAGAAGTTGAGAGTGGCATTTCAACTATTACACAATGCCTTGGCGAATTACCATTTTACTCTGTTTCTGAGGAGTCTGAGCCAACTGAGACATCAAAGAAGATTCAGCCTACCTCTTCTGCCATGGTGTCCTCTAGAAAGCCAGTTATTCTTGCTGATGGAACTTATGCTACACAAAGCGCAGCCTCTGAAACCACATTCTCCTCGCCTACAGTTGTTCAAGGATCACTGACTTCTGGAAATTTGAGGGCACTCCTTCTAACTGGTGATTTTTTCCTCGGAGCTGTGGTTGCTTGCACGTTGACCAAACTTGTTCTTAGGTTGGAAGAGGTTCAGTCTTCCAAAACTGAAGTAAACAAGACAGTATCACAGGCTTTGCTAATCATGGTTTCTATTTTGCAACTTGGGCAATCTCCTGTTTCTCCACACCCTATTGATAATGATTCGTATGAGCGGATTATGTTGTGCATAAAATTGCTTTGCCATAGGAATGTTGAGATGAAAAAGATATGGTTGGAATCCTGCCGCCAGAGTTTTGTCAAGATGATTTCTGAAAAACAGCTTAGAGAGATGGAGGAACTGAAGGCAAAGACCCAAACAACTCATGCTCAACCGGATGATCTAATTGACTTCTTCCATCTAAAGAGTCGGAAGGGAATGAGTCAACTTGAGTTGGAAGACCAGGTACAAGATGACCTAAAGCGTGCAACTGGAGAATTCACCAAGGACGAGAACGATGCTAACAAACTTAACCGCATTCTTCAACTCACAGGATTCAGTGACCCAGTCTATGCTGAAGCATATGTAACGGTACACCATTATGATATTGCTCTTGAAGTTACAGTAATCAACCGAACCAAGGAAACCCTTCAGAACTTGTGCTTGGAGTTAGCAACCATGGGTGATCTCAAACTTGTTGAGCGTCCTCAGAACTATAGTCTGGCACCTGAAAGAAGCATGCAGATTAAAGCAAACATCAAGGTCTCGTCCACAGAGACAGGAGTCATATTCGGGAACATCGTCTATGAGACATCAAATGTAATGGAGCGCAATGTTGTGGTTCTTAACGACATACACATTGATATCATGGACTATATCTCCCCTGCTGTGTGCTCAGAGGTTGCTTTCAGAACTATGTGGGCAGAGTTTGAATGGGAAAACAAGGTTGCTGTGAACACCACAATTCAAAACGAAAGAGAATTCCTCGACCACATTATCAAATCCACAAACATGAAATGTCTCACTGCTCCATCTGCAATAGCAGGTGAATGTGGATTCCTTGCAGCAAACTTATATGCAAAAAGTGTATTTGGTGAGGATGCTCTTGTGAATTTGAGTATTGAGAAGCAAACGGATGGAACATTGAGTGGTTACATAAGGATAAGGAGCAAGACGCAAGGGATTGCTCTAAGTCTTGGAGACAAAATCACCCTCAAACAAAAGGGTGGTAGCTGA SEQ ID NO: 68,Deduced amino acid sequence of the open reading frame of OO-6MDKSSTMLVHYDKGTPAVANEIKEALEGNDVEAKVDAMKKAIMLLLNGETIPQLFITIIRYVLPSEDHTIQKLLLLYLELIEKTDSKGKVLPEMILICQNLRNNLQHPNEYIRGVTLRFLCRMKETEIVEPLTPSVLQNLEHRHPFVRRNAILAIMSIYKLPHGDQLFVDAPEMIEKVLSTEQDPSAKRNAFLMLFTCAEERAVNYLLSNVDKVSDWNESLQMVVLELIRSVCKTKPAEKGKYIKIIISLLSATSSAVIYECAGTLVSLSSAPTAIRAAANTYCQLLLSQSDNNVKLILLDRLYELKTLHRDIMVELIIDVLRALSSPNLDIRRKTLDIALDLITHHNINEVVQMLKKEVVKTQSGELEKNGEYRQMLIQAIHACAVKFPEVASTVVHLLMDFLGDSNVASALDVVVFVREIIETNPKLRVSIITRLLDTFYQIRAGKVCPCALWIIGEYCLSLSEVESGISTITQCLGELPFYSVSEESEPTETSKKIQPTSSAMVSSRKPVILADGTYATQSAASETTFSSPTVVQGSLTSGNLRALLLTGDFFLGAVVACTLTKLVLRLEEVQSSKTEVNKTVSQALLIMVSILQLGQSPVSPHPIDNDSYERIMLCIKLLCHRNVEMKKIWLESCRQSFVKMISEKQLREMEELKAKTQTTHAQPDDLIDFFHLKSRKGMSQLELEDQVQDDLKRATGEFTKDENDANKLNRILQLTGFSDPVYAEAYVTVHHYDIALEVTVINRTKETLQNLCLELATMGDLKLVERPQNYSLAPERSMQIKANIKVSSTETGVIFGNIVYETSNVMERNVVVLNDIHIDIMDYISPAVCSEVAFRTMWAEFEWENKVAVNTTIQNEREFLDHIIKSTNMKCLTAPSAIAGECGFLAANLYAKSVFGEDALVNLSIEKQTDGTLSGYIRIRSKTQGIALSLGDKITLKQKGGS SEQ ID NO: 69, Nucleotide sequence of the open readingframe of OO-8 ATGGCGAAATCTCAGATCTGGTTTGGTTTTGCGTTACTCGCGTTGCTTCTGGTTTCAGCCGTAGCTGACGATGTGGTTGTTTTGACTGACGATAGCTTCGAAAAGGAAGTTGGTAAAGATAAAGGAGCTCTCGTCGAGTTTTACGCTCCCTGGTGTGGTCACTGCAAGAAACTTGCTCCAGAGTATGAAAAGCTAGGGGCAAGCTTCAAGAAGGCTAAGTCTGTGTTGATTGCAAAGGTTGATTGTGATGAGCAAAAGAGTGTCTGTACTAAATATGGTGTTAGTGGATACCCAACCATTCAGTGGTTTCCTAAAGGATCTCTTGAACCTCAAAAGTATGAGGGTCCACGCAATGCTGAAGCTTTGGCTGAATACGTGAACAAGGAAGGAGGCACCAACGTAAAATTAGCTGCAGTTCCACAAAACGTGGTTGTTTTGACACCTGACAATTTCGATGAGATTGTTCTGGATCAAAACAAAGATGTCCTAGTCGAATTTTATGCACCATGGTGTGGCCACTGCAAATCACTCGCTCCCACATACGAAAAGGTAGCCACAGTGTTTAAACAGGAAGAAGGTGTAGTCATCGCCAATTTGGATGCTGATGCACACAAAGCCCTTGGCGAGAAATATGGAGTGAGTGGATTCCCAACATTGAAATTCTTCCCAAAGGACAACAAAGCTGGTCACGATTATGACGGTGGCAGGGATTTAGATGACTTTGTAAGCTTCATCAACGAGAAATCTGGGACCAGCAGGGACAGTAAAGGGCAGCTTACTTCAAAGGCTGGTATAGTCGAAAGCTTAGATGCTTTGGTAAAAGAGTTAGTTGCAGCTAGTGAAGATGAGAAGAAGGCAGTGTTGTCTCGCATAGAAGAGGAAGCAAGTACCCTTAAGGGCTCCACCACGAGGTATGGAAAGCTTTACTTGAAACTCGCAAAGAGCTACATAGAAAAAGGTTCAGACTATGCTAGCAAAGAAACGGAGAGGCTTGGACGGGTGCTTGGGAAGTCGATAAGTCCAGTGAAAGCTGATGAACTCACTCTCAAGAGAAATATCCTAACCACGTTCGTTGCTTCTTCTTAA SEQ ID NO: 70,Deduced amino acid sequence of the open reading frame of OO-8MAKSQIWFGFALLALLLVSAVADDVVVLTDDSFEKEVGKDKGALVEFYAPWCGHCKKLAPEYEKLGASFKKAKSVLIAKVDCDEQKSVCTKYGVSGYPTIQWFPKGSLEPQKYEGPRNAEALAEYVNKEGGTNVKLAAVPQNVVVLTPDNFDEIVLDQNKDVLVEFYAPWCGHCKSLAPTYEKVATVFKQEEGVVIANLDADAHKALGEKYGVSGFPTLKFFPKDNKAGHDYDGGRDLDDFVSFINEKSGTSRDSKGQLTSKAGIVESLDALVKELVAASEDEKKAVLSRIEEEASTLKGSTTRYGKLYLKLAKSYIEKGSDYASKETERLGRVLGKSISPVKADELTLKRNILTTFVASS SEQ ID NO: 71, Nucleotide sequence of the openreading frame of OO-9ATGGCGTCGAGCGATGAGCGTCCAGGAGCGTATCCGGCACGTGACGGATCAGAGAACTTACCTCCGGGAGATCCAAAGACGATGAAGACGGTGGTGATGGATAAAGGAGCGGCGATGATGCAATCGTTGAAACCGATCAAACAGATGAGTCTCCATTTGTGTTCTTTCGCTTGTTATGGTCACGATCCTAGCCGTCAGATTGAAGTCAACTTCTATGTTCATCGACTCAACCAAGACTTTCTTCAATGTGCTGTTTACGATTGCGACTCCTCTAAACCCCATCTCATCGGGATCGAGTATATTGTGTCGGAGAGGTTATTTGAGAGTCTTGATCCGGAGGAGCAAAAGCTTTGGCACTCTCATGACTATGAGATCCAAACAGGCCTTCTAGTAACTCCAAGGGTCCCTGAGCTTGTAGCTAAGACAGAGCTTGAAAATATTGCCAAAACTTATGGGAAGTTTTGGTGCACTTGGCAGACCGATCGCGGGGATAAATTGCCACTTGGTGCACCATCACTTATGATGTCACCACAAGACGTGAATATGGGAAAGATCAAGCCAGGGCTATTGAAGAAACGTGACGATGAGTATGGAATCTCGACGGAATCTTTGAAGACGTCTCGAGTTGGAATTATGGGACCGGAGAAGAAAAATTCGATGGCTGATTATTGGGTTCATCACGGAAAAGGATTAGCGGTTGACATAATCGAAACTGAGATGCAGAAATTGGCTCCGTTCCCGTAA SEQ ID NO: 72, Deduced amino acidsequence of the open reading frame of OO-9MASSDERPGAYPARDGSENLPPGDPKTMKTVVMDKGAAMMQSLKPIKQMSLHLCSFACYGHDPSRQIEVNFYVHRLNQDFLQCAVYDCDSSKPHLIGIEYIVSERLFESLDPEEQKLWHSHDYEIQTGLLVTPRVPELVAKTELENIAKTYGKFWCTWQTDRGDKLPLGAPSLMMSPQDVNMGKIKPGLLKKRDDEYGISTESLKTSRVGIMGPEKKNSMADYWVHHGKGLAVDIIETEMQKLAPFP SEQ ID NO: 73, Nucleotide sequence of the openreading frame of OO-10ATGGCGACTCTTAAGGTTTCTGATTCTGTTCCTGCTCCTTCTGATGATGCTGAGCAATTGAGAACCGCTTTTGAAGGATGGGGTACGAACGAGGACTTGATCATATCAATCTTGGCTCACAGAAGTGCTGAACAGAGGAAAGTCATCAGGCAAGCATACCACGAAACCTACGGCGAAGACCTTCTCAAGACTCTTGACAAGGAGCTCTCTAACGATTTCGAGAGAGCTATCTTGTTGTGGACTCTTGAACCCGGTGAGCGTGATGCTTTATTGGCTAATGAAGCTACAAAAAGATGGACTTCAAGCAACCAAGTTCTTATGGAAGTTGCTTGCACAAGGACATCAACGCAGCTGCTTCACGCTAGGCAAGCTTACCATGCTCGCTACAAGAAGTCTCTTGAAGAGGACGTTGCTCACCACACTACCGGTGACTTCAGAAAGCTTTTGGTTTCTCTTGTTACCTCATACAGGTACGAAGGAGATGAAGTGAACATGACATTGGCTAAGCAAGAAGCTAAGCTGGTCCATGAGAAAATCAAGGACAAGCACTACAATGATGAGGATGTTATTAGAATCTTGTCCACAAGAAGCAAAGCTCAGATCAATGCTACTTTTAACCGTTACCAAGATGATCATGGCGAGGAAATTCTCAAGAGTCTTGAGGAAGGAGATGATGATGACAAGTTCCTTGCACTTTTGAGGTCAACCATTCAGTGCTTGACAAGACCAGAGCTTTACTTTGTCGATGTTCTTCGTTCAGCAATCAACAAAACTGGAACTGATGAAGGAGCACTCACTAGAATTGTGACCACAAGAGCTGAGATTGACTTGAAGGTCATTGGAGAGGAGTACCAGCGCAGGAACAGCATTCCTTTGGAGAAAGCTATTACCAAAGACACTCGTGGAGATTACGAGAAGATGCTCGTCGCACTTCTCGGTGAAGATGATGCTTAA SEQ ID NO: 74, Deduced amino acid sequence ofthe open reading frame of OO-10MATLKVSDSVPAPSDDAEQLRTAFEGWGTNEDLIISILAHRSAEQRKVIRQAYHETYGEDLLKTLDKELSNDFERAILLWTLEPGERDALLANEATKRWTSSNQVLMEVACTRTSTQLLHARQAYHARYKKSLEEDVAHHTTGDFRKLLVSLVTSYRYEGDEVNMTLAKQEAKLVHEKIKDKHYNDEDVIRILSTRSKAQINATFNRYQDDHGEEILKSLEEGDDDDKFLALLRSTIQCLTRPELYFVDVLRSAINKTGTDEGALTRIVTTRAEIDLKVIGEEYQRRNSIPLEKAITKDTRGDYEKMLVALLGEDDA SEQ ID NO: 75, Nucleotide sequence ofthe open reading frame of OO-11ATGGTGGATCTATTGAACTCGGTGATGAACCTGGTGGCGCCTCCAGCGACCATGGTGGTGATGGCCTTTGCATGGCCATTACTGTCTTTCATTAGCTTCTCCGAACGGGCTTACAACTCTTATTTCGCCACCGAAAATATGGAAGATAAAGTAGTTGTCATCACCGGAGCTTCATCGGCCATTGGAGAGCAAATAGCATATGAATATGCAAAAAGAGGAGCGAATTTGGTGTTGGTGGCGAGGAGAGAGCAGAGACTGAGAGTTGTGAGTAATAAGGCTAAACAGATTGGAGCCAACCATGTGATCATCATCGCTGCTGATGTCATCAAAGAAGATGACTGCCGCCGTTTTATCACCCAAGCCGTCAACTATTACGGCCGCGTGGATCATCTAGTGAATACAGCGAGTCTTGGACACACTTTTTACTTTGAGGAAGTGAGTGACACGACTGTGTTTCCACATTTGCTGGACATAAACTTCTGGGGGAATGTTTATCCGACATACGTAGCGTTGCCATACCTTCACCAGACGAATGGCCGAATAGTCGTGAATGCATCGGTTGAAAACTGGTTGCCTCTACCACGGATGAGTCTTTATTCTGCTGCAAAAGCAGCATTAGTCAACTTCTATGAGACGCTGCGTTTCGAGCTAAATGGAGACGTTGGTATAACTATCGCGACTCACGGGTGGATTGGCAGTGAGATGAGTGGAGGAAAGTTCATGCTAGAAGAAGGTGCTGAGATGCAATGGAAGGAAGAGAGAGAAGTACCTGCAAATGGTGGACCGCTAGAGGAATTTGCAAAGATGATTGTGGCAGGAGCTTGTAGGGGAGATGCATATGTGAAGTTTCCAAACTGGTACGATGTCTTTCTCCTCTATCGAGTCTTCACACCGAATGTACTGAGATGGACATTCAAGTTGTTACTGTCTACTGAGGGTACACGTAGAAGCTCCCTTGTTGGGGTCGGGTCAGGTATGCCTGTGGATGAATCCTCTTCACAAATGAAACTTATGCTTGAAGGAGGACCACCTCGAGTTCCTGCAAGCCCACCTAGGTATACCGCAAGCCCACCTCATTATACCGCAAGCCCACCACGGTATCCTGCAAGCCCACCTCGGTATCCTGCGAGCCCACCTCGGTTTTCACAGTTTAATATCCAAGAGTTGTAA SEQ ID NO: 76, Deduced amino acid sequence of theopen reading frame of OO-11MVDLLNSVMNLVAPPATMVVMAFAWPLLSFISFSERAYNSYFATENMEDKVVVITGASSAIGEQIAYEYAKRGANLVLVARREQRLRVVSNKAKQIGANHVIIIAADVIKEDDCRRFITQAVNYYGRVDHLVNTASLGHTFYFEEVSDTTVFPHLLDINFWGNVYPTYVALPYLHQTNGRIVVNASVENWLPLPRMSLYSAAKAALVNFYETLRFELNGDVGITIATHGWIGSEMSGGKFMLEEGAEMQWKEEREVPANGGPLEEFAKMIVAGACRGDAYVKFPNWYDVFLLYRVFTPNVLRWTFKLLLSTEGTRRSSLVGVGSGMPVDESSSQMKLMLEGGPPRVPASPPRYTASPPHYTASPPRYPASPPRYPASPPRFSQFNIQEL SEQ ID NO: 77,Nucleotide sequence of the open reading frame of OO-12ATGGCTGGAAAACTCATGCACGCTCTTCAGTACAACTCTTACGGTGGTGGCGCCGCCGGATTAGAGCATGTTCAAGTTCCGGTTCCAACACCAAAGAGTAATGAGGTTTGCCTGAAATTAGAAGCTACTAGTCTAAACCCTGTTGATTGGAAAATTCAGAAAGGAATGATCCGCCCATTTCTGCCCCGCAAGTTCCCCTGCATTCCAGCTACTGATGTTGCTGGAGAGGTCGTTGAGGTTGGATCAGGAGTAAAAAATTTTAAGGCTGGTGACAAAGTTGTAGCGGTTCTTAGCCATCTAGGTGGAGGTGGACTTGCTGAGTTCGCTGTTGCAACCGAGAAGCTGACTGTCAAAAGACCTCAAGAAGTGGGAGCAGCTGAAGCAGCAGCTTTACCTGTGGCGGGTCTAACCGCTCTCCAAGCTCTTACTAATCCTGCGGGGTTGAAGCTGGATGGTACAGGCAAGAAGGCGAACATCCTGGTCACAGCAGCATCTGGTGGGGTTGGTCACTATGCAGTCCAGCTGGCAAAACTTGCAAATGCTCACGTAACCGCTACATGTGGTGCCCGGAACATAGAGTTTGTCAAATCGTTGGGAGCGGATGAGGTTCTCGACTACAAGACTCCCGAGGGAGCCGCCCTCAAGAGTCCGTCGGGTAAAAAATATGACGCTGTGGTCCATTGTGCAAACGGGATTCCATTTTCGGTATTCGAACCAAATTTGTCGGAAAACGGGAAGGTGATAGACATCACACCGGGGCCTAATGCAATGTGGACTTATGCGGTTAAGAAAATAACCATGTCAAAGAAGCAGTTAGTGCCACTCTTGTTGATCCCAAAAGCTGAGAATTTGGAGTTTATGGTGAATCTAGTGAAAGAAGGGAAAGTGAAGACAGTGATTGACTCAAAGCATCCTTTGAGCAAAGCGGAGGATGCTTGGGCCAAAAGTATCGATGGTCATGCTACTGGGAAGATCATTGTC GAGCCATAA SEQ IDNO: 78, Deduced amino acid sequence of the open reading frame of OO-12MAGKLMHALQYNSYGGGAAGLEHVQVPVPTPKSNEVCLKLEATSLNPVDWKIQKGMIRPFLPRKFPCIPATDVAGEVVEVGSGVKNFKAGDKVVAVLSHLGGGGLAEFAVATEKLTVKRPQEVGAAEAAALPVAGLTALQALTNPAGLKLDGTGKKANILVTAASGGVGHYAVQLAKLANAHVTATCGARNIEFVKSLGADEVLDYKTPEGAALKSPSGKKYDAVVHCANGIPFSVFEPNLSENGKVIDITPGPNAMWTYAVKKITMSKKQLVPLLLIPKAENLEFMVNLVKEGKVKTVIDSKHPLSKAEDAWAKSIDGHATGKIIVEP SEQ ID NO: 79,Nucleotide sequence of the open reading frame of pp82ATGGAAATTCCCTTAGGTCGAGATGGCGAGGGTATGCAGTCAAAGCAGTGCCCGCGCGGCCACTGGCGTCCAGCGGAAGACGACAAGCTGCGAGAACTAGTGTCCCAGTTTGGACCTCAAAACTGGAATCTCATAGCAGAGAAACTTCAGGGTCGATCAGGGAAAAGCTGCAGGCTACGGTGGTTCAATCAGCTGGACCCTCGCATCAACCGGCACCCATTCTCGGAAGAAGAGGAAGAGCGGCTGCTTATAGCACACAAGCGCTACGGCAACAAGTGGGCATTGATCGCGCGCCTCTTTCCGGGCCGCACAGACAACGCGGTGAAGAATCACTGGCACGTTGTGACGGCAAGACAGTCCCGTGAACGGACACGAACTTACGGCCGTATCAAAGGTCCGGTACATCGAAGAGGCAAGGGTAACCGTATCAATACCTCCGCACTTGGAAATTACCATCACGATTCGAAGGGAGCTCTCACAGCCTGGATTGAGTCGAAGTATGCGACAGTCGAGCAGTCTGCGGAAGGGCTCGCTAGGTCTCCTTGTACCGGCAGAGGCTCTCCTCCTCTACCCACCGGTTTCAGTATACCGCAGATTTCCGGCGGCGCCTTCCATCGACCGACAAACATGAGTACTAGTCCTCTTAGCGATGTGACTATCGAGTCGCCAAAGTTTAGCAACTCCGAAAATGCGCAAATAATAACCGCGCCCGTCCTGCAAAAGCCAATGGGAGATCCCAGGTCAGTATGCTTGCCGAATTCGACTGTTTCCGACAAGCAGCAAGTGCTGCAGAGTAATTCCATCGACGGTCAGATCTCCTCCGGGCTCCAGACAAGCGCAATAGTAGCGCATGATGAGAAATCGGGCGTCATTTCAATGAATCATCAAGCACCGGATATGTCCTGTGTTGGATTGAAGTCAAATTTTCAGGGGAGTCTCCATCCTGGCGCTGTTAGATCTTCTTGGAATCAATCCCTTCCCCACTGTTTTGGCCACAGTAACAAGTTGGTGGAGGAGTGCAGGAGTTCTACAGGCGCATGCACTGAACGCTCTGAGATTCTGCAAGAACAGCATTCTAGCCTTCAGTTTAAATGCAGCACTGCGTACAATACTGGAAGATATCAACATGAAAACCTTTGTGGGCCAGCATTCTCGCAACAAGACACAGCGAACGAGGTTGCGAATTTTTCTACGTTGGCATTCTCCGGCCTAGTGAAGCATCGCCAAGAGAGGTTGTGCAAAGATAGTGGATCTGCTCTCAAGCTGGGACTATCATGGGTTACATCCGATAGCACTCTTGACTTGAGTGTTGCCAAAATGTCAGCATCGCAGCCAGAGCAGTCTGCGCCGGTTGCATTCATTGATTTTCTAGGCGTGGGAGCGGCCTGA SEQ ID NO: 80, Deduced amino acid sequenceof the open reading frame of pp82MEIPLGRDGEGMQSKQCPRGHWRPAEDDKLRELVSQFGPQNWNLIAEKLQGRSGKSCRLRWFNQLDPRINRHPFSEEEEERLLIAHKRYGNKWALIARLFPGRTDNAVKNHWHVVTARQSRERTRTYGRIKGPVHRRGKGNRINTSALGNYHHDSKGALTAWIESKYATVEQSAEGLARSPCTGRGSPPLPTGFSIPQISGGAFHRPTNMSTSPLSDVTIESPKFSNSENAQIITAPVLQKPMGDPRSVCLPNSTVSDKQQVLQSNSIDGQISSGLQTSAIVAHDEKSGVISMNHQAPDMSCVGLKSNFQGSLHPGAVRSSWNQSLPHCFGHSNKLVEECRSSTGACTERSEILQEQHSSLQFKCSTAYNTGRYQHENLCGPAFSQQDTANEVANFSTLAFSGLVKHRQERLCKDSGSALKLGLSWVTSDSTLDLSVAKMSASQPEQSAPVAFIDF LGVGAA SEQ IDNO: 81, Nucleotide sequence of the open reading frame of Pk225ATGGAGATGAACATTAAGTTTCCAGTTATAGACTTGTCTAAGCTCAATGGTGAAGAGAGAGACCAAACCATGGCTTTGATCGACGATGCTTGTCAAAACTGGGGCTTCTTCGAGCTGGTGAACCATGGACTACCATATGATCTAATGGACAACATTGAGAGGATGACAAAGGAACACTACAAGAAACATATGGAACAAAAGTTCAAAGAAATGCTTCGTTCCAAAGGTTTAGATACCCTCGAGACCGAAGTTGAAGATGTCGATTGGGAAAGCACTTTCTACCTCCATCATCTCCCTCAATCTAACCTATACGACATCCCTGATATGTCAAATGAATACCGATTGGCAATGAAGGATTTTGGGAAGAGGCTTGAGATTCTAGCTGAAGAGCTATTGGACTTGTTGTGTGAGAATCTAGGGTTGGAGAAAGGGTACTTGAAGAAGGTGTTTCATGGGACAACGGGTCCAACTTTTGCGACAAAGCTTAGCAACTATCCACCATGTCCTAAACCAGAGATGATCAAAGGGCTTAGGGCTCACACAGATGCAGGAGGCCTCATTTTGCTGTTTCAAGATGATAAGGTCAGTGGTCTCCAGCTTCTTAAAGATGGTGATTGGGTTGATGTTCCTCCTCTCAAGCATTCCATTGTCATCAACCTTGGTGACCAACTTGAGGTGATAACAAACGGGAAGTACAAGAGTGTAATGCACCGTGTGATGACCCAGAAAGAAGGAAACAGGATGTCTATCGCGTCGTTTTACAACCCCGGAAGCGATGCTGAGATCTCTCCGGCAACATCTCTTGTGGATAAAGACTCAAAATACCCAAGCTTTGTGTTTGATGACTACATGAAACTCTATGCCGGACTCAAGTTTCAGGCCAAGGAGCCACGGTTCGAGGCGATGAAAAATGCTGAAGCAGCTGCGGATTTGAATCCGGTGGCTGTGGTTGAGACATTCTAA SEQ ID NO: 82, Deduced amino acidsequence of the open reading frame of Pk225MEMNIKFPVIDLSKLNGEERDQTMALIDDACQNWGFFELVNHGLPYDLMDNIERMTKEHYKKHMEQKFKEMLRSKGLDTLETEVEDVDWESTFYLHHLPQSNLYDIPDMSNEYRLAMKDFGKRLEILAEELLDLLCENLGLEKGYLKKVFHGTTGPTFATKLSNYPPCPKPEMIKGLRAHTDAGGLILLFQDDKVSGLQLLKDGDWVDVPPLKHSIVINLGDQLEVITNGKYKSVMHRVMTQKEGNRMSIASFYNPGSDAEISPATSLVDKDSKYPSFVFDDYMKLYAGLKFQAKEPRFEAMKNAEAAADLNPVAVVETF

1. An isolated lipid metabolism protein (LMP) nucleic acid comprising apolynucleotide sequence encoding a polypeptide that functions as amodulator of a seed storage compound in a plant, wherein thepolynucleotide sequence is selected from the group consisting of: a) apolynucleotide sequence as defined in SEQ ID NO: 79; b) a polynucleotidesequence encoding a polypeptide as defined in SEQ ID NO: 80; c) apolynucleotide sequence comprising at least 60 consecutive nucleotidesof a) or b) above; d) a polynucleotide sequence encoding a polypeptidehaving at least 70% sequence identity with the polynucleotide sequenceof a) or b) above; e) a polynucleotide sequence encoding a polypeptidehaving at least 70% identity to the amino acid sequence of SEQ ID NO:80; f) a polynucleotide sequence complementary to the polynucleotidesequence of a) or b) above; and g) a polynucleotide sequence thathybridizes to the complement of the full-length nucleic acid of a) or b)above under stringent conditions of 6× sodium chloride/sodium citrate(SSC) at 65° C. followed by one or more washes in 0.2×SSC at 50 to 65°C.
 2. The isolated nucleic acid of claim 1, wherein the nucleic acidcomprises the polynucleotide sequence of SEQ ID NO: 79 or apolynucleotide sequence which encodes the polypeptide sequence of SEQ IDNO:
 80. 3. The isolated nucleic acid of claim 1, wherein thepolynucleotide sequence encodes a polypeptide having at least 90%sequence identity with the sequence of SEQ ID NO:
 80. 4. The isolatednucleic acid of claim 1, wherein the polynucleotide sequence encodes apolypeptide having at least 95% sequence identity with the sequence ofSEQ ID NO:
 80. 5. An expression vector comprising the isolated nucleicacid of claim
 1. 6. The expression vector of claim 5, wherein thenucleic acid is operatively linked to a heterologous promoter selectedfrom the group consisting of a seed-specific promoter, a root-specificpromoter, and a non-tissue-specific promoter.
 7. A method of producing atransgenic plant having a modified level of a seed storage compound ascompared to a corresponding wild type variety of the plant comprising,transforming a plant cell with an expression vector comprising a lipidmetabolism protein (LMP) nucleic acid and generating from the plant cellthe transgenic plant, wherein the nucleic acid encodes a polypeptidethat functions as a modulator of a seed storage compound in the plant,and wherein the nucleic acid comprises the LMP nucleic acid of claim 1.8. The method of claim 7, wherein the LMP nucleic acid comprises thepolynucleotide sequence of SEQ ID NO:
 79. 9. The method of claim 7,wherein the LMP nucleic acid comprises a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:
 80. 10. The method of claim 7,wherein the level of a seed storage compound is increased in thetransgenic plant as compared to a corresponding wild type plant.
 11. Themethod of claim 7, wherein the LMP nucleic acid is operatively linked toa heterologous promoter selected from the group consisting of aseed-specific promoter, a root-specific promoter, and anon-tissue-specific promoter.
 12. The method of claim 7, wherein themodified level of the seed storage compound is due to the overexpressionor down-regulation of the LMP nucleic acid.
 13. The method of claim 7,wherein the LMP nucleic acid comprises a polynucleotide having at least90% sequence identity to the nucleic acid of SEQ ID NO: 79 or apolynucleotide sequence encoding a polypeptide having at least 90%identity to the amino acid sequence of SEQ ID NO:
 80. 14. The method ofclaim 7, wherein the nucleic acid encodes a polypeptide that contains alipid metabolism domain.
 15. The method of claim 14, wherein the nucleicacid encodes a polypeptide comprising the sequence of SEQ ID NO:
 80. 16.A transgenic plant made by the method of claim 7, wherein expression ofthe LMP nucleic acid in the plant results in a modified level of a seedstorage compound in the plant as compared to a corresponding wild typevariety of the plant.
 17. A transgenic plant or part thereof comprisingthe isolated LMP nucleic acid of claim
 1. 18. The transgenic plant ofclaim 17, wherein the plant is a dicotyledonous plant.
 19. Thetransgenic plant of claim 17, wherein the plant is a monocotyledonousplant.
 20. The transgenic plant of claim 17, wherein the plant is an oilproducing species.
 21. The transgenic plant of claim 17, wherein theplant is selected from the group consisting of rapeseed, canola,linseed, soybean, sunflower, maize, oat, rye, barley, wheat, sugarbeet,tagetes, cotton, oil palm, coconut palm, flax, castor, and peanut. 22.The transgenic plant of claim 17, wherein the level of the seed storagecompound is increased in the transgenic plant as compared to acorresponding wild type variety of the plant.
 23. The transgenic plantof claim 17, wherein the seed storage compound is selected from thegroup consisting of a lipid, a fatty acid, a starch, and a seed storageprotein.
 24. A seed produced by the transgenic plant of claim 17,wherein the plant expresses the LMP polypeptide and wherein the plant istrue breeding for a modified level of the seed storage compound ascompared to a corresponding wild type variety of the plant.
 25. Thetransgenic plant or part thereof of claim 17, wherein the part thereofcomprises a seed.